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Sample records for quantitative phosphoproteomics dissection

  1. Quantitative Phosphoproteomic Analysis of T-Cell Receptor Signaling.

    PubMed

    Ahsan, Nagib; Salomon, Arthur R

    2017-01-01

    TCR signaling critically depends on protein phosphorylation across many proteins. Localization of each phosphorylation event relative to the T-cell receptor (TCR) and canonical T-cell signaling proteins will provide clues about the structure of TCR signaling networks. Quantitative phosphoproteomic analysis by mass spectrometry provides a wide-scale view of cellular phosphorylation networks. However, analysis of phosphorylation by mass spectrometry is still challenging due to the relative low abundance of phosphorylated proteins relative to all proteins and the extraordinary diversity of phosphorylation sites across the proteome. Highly selective enrichment of phosphorylated peptides is essential to provide the most comprehensive view of the phosphoproteome. Optimization of phosphopeptide enrichment methods coupled with highly sensitive mass spectrometry workflows significantly improves the sequencing depth of the phosphoproteome to over 10,000 unique phosphorylation sites from complex cell lysates. Here we describe a step-by-step method for phosphoproteomic analysis that has achieved widespread success for identification of serine, threonine, and tyrosine phosphorylation. Reproducible quantification of relative phosphopeptide abundance is provided by intensity-based label-free quantitation. An ideal set of mass spectrometry analysis parameters is also provided that optimize the yield of identified sites. We also provide guidelines for the bioinformatic analysis of this type of data to assess the quality of the data and to comply with proteomic data reporting requirements.

  2. Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

    PubMed Central

    Jacobs, Kris; Klammer, Martin; Jordan, Nicole; Elschenbroich, Sarah; Parade, Marc; Jacoby, Edgar; Linders, Joannes T. M.; Brehmer, Dirk; Cools, Jan; Daub, Henrik

    2016-01-01

    The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies. PMID:26745281

  3. Quantitative label-free phosphoproteomics strategy for multifaceted experimental designs.

    PubMed

    Soderblom, Erik J; Philipp, Melanie; Thompson, J Will; Caron, Marc G; Moseley, M Arthur

    2011-05-15

    Protein phosphorylation is a critical regulator of signaling in nearly all eukaryotic cellular pathways and dysregulated phosphorylation has been implicated in an array of diseases. The majority of MS-based quantitative phosphorylation studies are currently performed from transformed cell lines because of the ability to generate large amounts of starting material with incorporated isotopically labeled amino acids during cell culture. Here we describe a general label-free quantitative phosphoproteomic strategy capable of directly analyzing relatively small amounts of virtually any biological matrix, including human tissue and biological fluids. The strategy utilizes a TiO(2) enrichment protocol in which the selectivity and recovery of phosphopeptides were optimized by assessing a twenty-point condition matrix of binding modifier concentrations and peptide-to-resin capacity ratios. The quantitative reproducibility of the TiO(2) enrichment was determined to be 16% RSD through replicate enrichments of a wild-type Danio rerio (zebrafish) lysate. Measured phosphopeptide fold-changes from alpha-casein spiked into wild-type zebrafish lysate backgrounds were within 5% of the theoretical value. Application to a morpholino induced knock-down of G protein-coupled receptor kinase 5 (GRK5) in zebrafish embryos resulted in the quantitation of 719 phosphorylated peptides corresponding to 449 phosphorylated proteins from 200 μg of zebrafish embryo lysates.

  4. The current state of the art of quantitative phosphoproteomics and its applications to diabetes research.

    PubMed

    Chan, Chi Yuet X'avia; Gritsenko, Marina A; Smith, Richard D; Qian, Wei-Jun

    2016-01-01

    Protein phosphorylation is a fundamental regulatory mechanism in many cellular processes and aberrant perturbation of phosphorylation has been implicated in various human diseases. Kinases and their cognate inhibitors have been considered as hotspots for drug development. Therefore, the emerging tools, which enable a system-wide quantitative profiling of phosphoproteome, would offer a powerful impetus in unveiling novel signaling pathways, drug targets and/or biomarkers for diseases of interest. This review highlights recent advances in phosphoproteomics, the current state of the art of the technologies and the challenges and future perspectives of this research area. Finally, some exemplary applications of phosphoproteomics in diabetes research are underscored.

  5. The current state of the art of quantitative phosphoproteomics and its applications to diabetes research

    SciTech Connect

    Chan, Chi Yuet X’avia; Gritsenko, Marina A.; Smith, Richard D.; Qian, Wei-Jun

    2016-03-17

    Protein phosphorylation is a fundamental regulatory mechanism in many cellular processes and aberrant perturbation of phosphorylation has been revealed in various human diseases. Kinases and their cognate inhibitors have been hotspot for drug development. Therefore, the emerging tools, which enable a system-wide quantitative profiling of phosphoproteome, would offer a powerful impetus in unveiling novel signaling pathways, drug targets and/or biomarkers for the disease of interest. In this review, we will highlight recent advances in phosphoproteomics, the current state-of-the-art of the technologies, and the challenges and future perspectives of this research area. Finally, we will underscore some exemplary applications of phosphoproteomics in diabetes research.

  6. Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis*

    PubMed Central

    Queiroz, Rayner M. L.; Charneau, Sébastien; Mandacaru, Samuel C.; Schwämmle, Veit; Lima, Beatriz D.; Roepstorff, Peter; Ricart, Carlos A. O.

    2014-01-01

    Chagas disease is a tropical neglected disease endemic in Latin America caused by the protozoan Trypanosoma cruzi. The parasite has four major life stages: epimastigote, metacyclic trypomastigote, bloodstream trypomastigote, and amastigote. The differentiation from infective trypomastigotes into replicative amastigotes, called amastigogenesis, takes place in vivo inside mammalian host cells after a period of incubation in an acidic phagolysosome. This differentiation process can be mimicked in vitro by incubating tissue-culture-derived trypomastigotes in acidic DMEM. Here we used this well-established differentiation protocol to perform a comprehensive quantitative proteomic and phosphoproteomic analysis of T. cruzi amastigogenesis. Samples from fully differentiated forms and two biologically relevant intermediate time points were Lys-C/trypsin digested, iTRAQ-labeled, and multiplexed. Subsequently, phosphopeptides were enriched using a TiO2 matrix. Non-phosphorylated peptides were fractionated via hydrophilic interaction liquid chromatography prior to LC-MS/MS analysis. LC-MS/MS and bioinformatics procedures were used for protein and phosphopeptide quantitation, identification, and phosphorylation site assignment. We were able to identify regulated proteins and pathways involved in coordinating amastigogenesis. We also observed that a significant proportion of the regulated proteins were membrane proteins. Modulated phosphorylation events coordinated by protein kinases and phosphatases that are part of the signaling cascade induced by incubation in acidic medium were also evinced. To our knowledge, this work is the most comprehensive quantitative proteomics study of T. cruzi amastigogenesis, and these data will serve as a trustworthy basis for future studies, and possibly for new potential drug targets. PMID:25225356

  7. Quantitative Proteomic and Phosphoproteomic Comparison of 2D and 3D Colon Cancer Cell Culture Models.

    PubMed

    Yue, Xiaoshan; Lukowski, Jessica K; Weaver, Eric M; Skube, Susan B; Hummon, Amanda B

    2016-12-02

    Cell cultures are widely used model systems. Some immortalized cell lines can be grown in either two-dimensional (2D) adherent monolayers or in three-dimensional (3D) multicellular aggregates, or spheroids. Here, the quantitative proteome and phosphoproteome of colon carcinoma HT29 cells cultures in 2D monolayers and 3D spheroids were compared with a stable isotope labeling of amino acids (SILAC) labeling strategy. Two biological replicates from each sample were examined, and notable differences in both the proteome and the phosphoproteome were determined by nanoliquid chromatography tandem mass spectrometry (LC-MS/MS) to assess how growth configuration affects molecular expression. A total of 5867 protein groups, including 2523 phosphoprotein groups and 8733 phosphopeptides were identified in the samples. The Gene Ontology analysis revealed enriched GO terms in the 3D samples for RNA binding, nucleic acid binding, enzyme binding, cytoskeletal protein binding, and histone binding for their molecular functions (MF) and in the process of cell cycle, cytoskeleton organization, and DNA metabolic process for the biological process (BP). The KEGG pathway analysis indicated that 3D cultures are enriched for oxidative phosphorylation pathways, metabolic pathways, peroxisome pathways, and biosynthesis of amino acids. In contrast, analysis of the phosphoproteomes indicated that 3D cultures have decreased phosphorylation correlating with slower growth rates and lower cell-to-extracellular matrix interactions. In sum, these results provide quantitative assessments of the effects on the proteome and phosphoproteome of culturing cells in 2D versus 3D cell culture configurations.

  8. Quantitative phosphoproteomics by mass spectrometry: Past, present, and future

    PubMed Central

    Nita-Lazar, Aleksandra; Saito-Benz, Hideshiro; White, Forest M.

    2009-01-01

    Protein phosphorylation-mediated signaling networks regulate much of the cellular response to external stimuli, and dysregulation in these networks has been linked to multiple disease states. Significant advancements have been made over the past decade to enable the analysis and quantification of cellular protein phosphorylation events, but comprehensive analysis of the phosphoproteome is still lacking, as is the ability to monitor signaling at the network level while comprehending the biological implications of each phosphorylation site. In this review we highlight many of the technological advances over the past decade and describe some of the latest applications of these tools to uncover signaling networks in a variety of biological settings. We finish with a concise discussion of the future of the field, including additional advances that are required to link protein phosphorylation analysis with biological insight. PMID:18846511

  9. Quantitative phospho-proteomics reveals the Plasmodium merozoite triggers pre-invasion host kinase modification of the red cell cytoskeleton.

    PubMed

    Zuccala, Elizabeth S; Satchwell, Timothy J; Angrisano, Fiona; Tan, Yan Hong; Wilson, Marieangela C; Heesom, Kate J; Baum, Jake

    2016-02-02

    The invasive blood-stage malaria parasite - the merozoite - induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture.

  10. Quantitative phosphoproteomic analysis of early seed development in rice (Oryza sativa L.).

    PubMed

    Qiu, Jiehua; Hou, Yuxuan; Tong, Xiaohong; Wang, Yifeng; Lin, Haiyan; Liu, Qing; Zhang, Wen; Li, Zhiyong; Nallamilli, Babi R; Zhang, Jian

    2016-02-01

    Rice (Oryza sativa L.) seed serves as a major food source for over half of the global population. Though it has been long recognized that phosphorylation plays an essential role in rice seed development, the phosphorylation events and dynamics in this process remain largely unknown so far. Here, we report the first large scale identification of rice seed phosphoproteins and phosphosites by using a quantitative phosphoproteomic approach. Thorough proteomic studies in pistils and seeds at 3, 7 days after pollination resulted in the successful identification of 3885, 4313 and 4135 phosphopeptides respectively. A total of 2487 proteins were differentially phosphorylated among the three stages, including Kip related protein 1, Rice basic leucine zipper factor 1, Rice prolamin box binding factor and numerous other master regulators of rice seed development. Moreover, differentially phosphorylated proteins may be extensively involved in the biosynthesis and signaling pathways of phytohormones such as auxin, gibberellin, abscisic acid and brassinosteroid. Our results strongly indicated that protein phosphorylation is a key mechanism regulating cell proliferation and enlargement, phytohormone biosynthesis and signaling, grain filling and grain quality during rice seed development. Overall, the current study enhanced our understanding of the rice phosphoproteome and shed novel insight into the regulatory mechanism of rice seed development.

  11. Quantitative Phosphoproteomics Identifies Filaggrin and other Targets of Ionizing Radiation in a Human Skin Model

    SciTech Connect

    Yang, Feng; Waters, Katrina M.; Webb-Robertson, Bobbie-Jo M.; Sowa, Marianne B.; Freiin von Neubeck, Claere H.; Aldrich, Joshua T.; Markillie, Lye Meng; Wirgau, Rachel M.; Gristenko, Marina A.; Zhao, Rui; Camp, David G.; Smith, Richard D.; Stenoien, David L.

    2012-04-17

    Our objective here was to perform a quantitative phosphoproteomic study on a reconstituted human skin tissue to identify low and high dose ionizing radiation dependent signaling in a complex 3-dimensional setting. Application of an isobaric labeling strategy using sham and 3 radiation doses (3, 10, 200 cGy) resulted in the identification of 1113 unique phosphopeptides. Statistical analyses identified 151 phosphopeptides showing significant changes in response to radiation and radiation dose. Proteins responsible for maintaining skin structural integrity including keratins and desmosomal proteins (desmoglein, desmoplakin, plakophilin 1 and 2,) had altered phosphorylation levels following exposure to both low and high doses of radiation. A phosphorylation site present in multiple copies in the linker regions of human profilaggrin underwent the largest fold change. Increased phosphorylation of these sites coincided with altered profilaggrin processing suggesting a role for linker phosphorylation in human profilaggrin regulation. These studies demonstrate that the reconstituted human skin system undergoes a coordinated response to ionizing radiation involving multiple layers of the stratified epithelium that serve to maintain skin barrier functions and minimize the damaging consequences of radiation exposure.

  12. Label-free quantitative phosphoproteomic profiling of cellular response induced by an insect cytokine paralytic peptide.

    PubMed

    Song, Liang; Wang, Fei; Dong, Zhaoming; Hua, Xiaoting; Xia, Qingyou

    2017-02-10

    Paralytic peptide (PP) participates in diverse physiological processes as an insect cytokine, such as immunity control, paralysis induction, regulation of cell morphology and proliferation. To investigate the molecular mechanism underlying those physiological activities, we systematically investigated the global phosphorylation events in fat body of silkworm larvae induced by PP through label-free quantitative phosphoproteomics. 2534 phosphosites were finally identified, of which the phosphorylation level of 620 phosphosites on 244 proteins was significantly up-regulated and 67 phosphosites on 43 proteins was down-regulated. Among those proteins, 13 were protein kinases (PKs), 13 were transcription factors (TFs) across 10 families and 17 were metabolism related enzymes. Meanwhile, Motif-X analysis of the phosphorylation sites showed that 16 motifs are significantly enriched, including 8 novel phosphorylation motifs. In addition, KEGG and functional interacting network analysis revealed that phosphorylation cascades play the crucial regulation roles in PP-dependent signaling pathways, and highlighted the potential central position of the mitogen-activated protein kinases (MAPKs) in them. These analyses provide direct insights into the molecule mechanisms of cellular response induced by PP.

  13. Quantitative Proteomic and Phosphoproteomic Approaches for Deciphering the Signaling Pathway for Tension Wood Formation in Poplar.

    PubMed

    Mauriat, Mélanie; Leplé, Jean-Charles; Claverol, Stéphane; Bartholomé, Jérôme; Negroni, Luc; Richet, Nicolas; Lalanne, Céline; Bonneu, Marc; Coutand, Catherine; Plomion, Christophe

    2015-08-07

    Trees adjust their growth following forced changes in orientation to re-establish a vertical position. In angiosperms, this adjustment involves the differential regulation of vascular cambial activity between the lower (opposite wood) and upper (tension wood) sides of the leaning stem. We investigated the molecular mechanisms leading to the formation of differential wood types through a quantitative proteomic and phosphoproteomic analysis on poplar subjected to a gravitropic stimulus. We identified and quantified 675 phosphopeptides, corresponding to 468 phosphoproteins, and 3 763 nonphosphorylated peptides, corresponding to 1 155 proteins, in the differentiating xylem of straight-growing trees (control) and trees subjected to a gravitational stimulus during 8 weeks. About 1% of the peptides were specific to a wood type (straight, opposite, or tension wood). Proteins quantified in more than one type of wood were more numerous: a mixed linear model showed 389 phosphopeptides and 556 proteins to differ in abundance between tension wood and opposite wood. Twenty-one percent of the phosphoproteins identified here were described in their phosphorylated form for the first time. Our analyses revealed remarkable developmental molecular plasticity, with wood type-specific phosphorylation events, and highlighted the involvement of different proteins in the biosynthesis of cell wall components during the formation of the three types of wood.

  14. Urinary proteomic and non-prefractionation quantitative phosphoproteomic analysis during pregnancy and non-pregnancy

    PubMed Central

    2013-01-01

    Background Progress in the fields of protein separation and identification technologies has accelerated research into biofluids proteomics for protein biomarker discovery. Urine has become an ideal and rich source of biomarkers in clinical proteomics. Here we performed a proteomic analysis of urine samples from pregnant and non-pregnant patients using gel electrophoresis and high-resolution mass spectrometry. Furthermore, we also apply a non-prefractionation quantitative phosphoproteomic approach using mTRAQ labeling to evaluate the expression of specific phosphoproteins during pregnancy comparison with non-pregnancy. Results In total, 2579 proteins (10429 unique peptides) were identified, including 1408 from the urine of pregnant volunteers and 1985 from the urine of non-pregnant volunteers. One thousand and twenty-three proteins were not reported in previous studies at the proteome level and were unique to our study. Furthermore, we obtained 237 phosphopeptides, representing 105 phosphoproteins. Among these phosphoproteins, 16 of them were found to be significantly differentially expressed, of which 14 were up-regulated and two were down-regulated in urine samples from women just before vaginal delivery. Conclusion Taken together, these results offer a comprehensive urinary proteomic profile of healthy women during before and after vaginal delivery and novel information on the phosphoproteins that are differentially regulated during the maintenance of normal pregnancy. Our results may provide a better understanding of the mechanisms of pregnancy maintenance, potentially leading to the development of biomarker-based sensitive assays for understanding pregnancy. PMID:24215720

  15. Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells

    PubMed Central

    Rusin, Scott F.; Schlosser, Kate A.; Adamo, Mark E.; Kettenbach, Arminja N.

    2017-01-01

    Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry–based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c–dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2–dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. PMID:26462736

  16. Quantitative Phosphoproteomics Reveals the Role of Protein Arginine Phosphorylation in the Bacterial Stress Response*

    PubMed Central

    Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

    2014-01-01

    Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response. PMID:24263382

  17. Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition

    PubMed Central

    Beck, Florian; Geiger, Jörg; Gambaryan, Stepan; Solari, Fiorella A.; Dell’Aica, Margherita; Loroch, Stefan; Mattheij, Nadine J.; Mindukshev, Igor; Pötz, Oliver; Jurk, Kerstin; Burkhart, Julia M.; Fufezan, Christian; Heemskerk, Johan W. M.; Walter, Ulrich

    2017-01-01

    Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein–coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin αIIbβ3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPase-activating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3ASer312, CALDAG-GEFISer587, ENSASer109), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways. PMID:28060719

  18. SPECHT – Single-stage phosphopeptide enrichment and stable-isotope chemical tagging: Quantitative phosphoproteomics of insulin action in muscle

    PubMed Central

    Kettenbach, Arminja N.; Sano, Hiroyuki; Keller, Susanna R.; Lienhard, Gustav E.; Gerber, Scott A.

    2014-01-01

    The study of cellular signaling remains a significant challenge for translational and clinical research. In particular, robust and accurate methods for quantitative phosphoproteomics in tissues and tumors represent significant hurdles for such efforts. In the present work, we design, implement and validate a method for single-stage phosphopeptide enrichment and stable isotope chemical tagging, or SPECHT, that enables the use of iTRAQ, TMT and/or reductive dimethyl-labeling strategies to be applied to phosphoproteomics experiments performed on primary tissue. We develop and validate our approach using reductive dimethyl-labeling and HeLa cells in culture, and find these results indistinguishable from data generated from more traditional SILAC-labeled HeLa cells mixed at the cell level. We apply the SPECHT approach to the quantitative analysis of insulin signaling in a murine myotube cell line and muscle tissue, identify known as well as new phosphorylation events, and validate these phosphorylation sites using phospho-specific antibodies. Taken together, our work validates chemical tagging post-single-stage phosphoenrichment as a general strategy for studying cellular signaling in primary tissues. PMID:25463755

  19. Integration of conventional quantitative and phospho-proteomics reveals new elements in activated Jurkat T-cell receptor pathway maintenance.

    PubMed

    Jouy, Florent; Müller, Stephan A; Wagner, Juliane; Otto, Wolfgang; von Bergen, Martin; Tomm, Janina M

    2015-01-01

    Recent years have seen a constant development of tools for the global assessment of phosphoproteins. Here, we outline a concept for integrating approaches for quantitative proteomics and phosphoproteomics. The strategy was applied to the analysis of changes in signalling and protein synthesis occurring after activation of the T-cell receptor (TCR) pathway in a T-cell line (Jurkat cells). For this purpose, peptides were obtained from four biological replicates of activated and control Jurkat T-cells and phosphopeptides enriched via a TiO2-based chromatographic step. Both phosphopeptide-enriched and flow-through fractions were analyzed by LC-MS. We observed 1314 phosphopeptides in the enriched fraction whereas 19 were detected in the flow-through, enabling the quantification of 414 and eight phosphoproteins in the respective fractions. Pathway analysis revealed the differential regulation of many metabolic pathways. Among the quantified proteins, 11 kinases with known TCR-related function were detected. A kinase-substrate database search for the phosphosites identified also confirmed the activity of a further ten kinases. In total, these two approaches provided evidence of 19 unique TCR-related kinases. The combination of phosphoproteomics and conventional quantitative shotgun analysis leads to a more comprehensive assessment of the signalling networks needed for the maintenance of the activated status of Jurkat T-cells.

  20. Quantitative Phosphoproteomics Reveals Novel Phosphorylation Events in Insulin Signaling Regulated by Protein Phosphatase 1 Regulatory Subunit 12A

    PubMed Central

    Zhang, Xiangmin; Ma, Danjun; Caruso, Michael; Lewis, Monique; Qi, Yue; Yi, Zhengping

    2014-01-01

    Serine/threonine protein phosphatase 1 regulatory subunit 12A (PPP1R12A) modulates the activity and specificity of the catalytic subunit of protein phosphatase 1, regulating various cellular processes via dephosphorylation. Nonetheless, little is known about phosphorylation events controlled by PPP1R12A in skeletal muscle insulin signaling. Here, we used quantitative phosphoproteomics to generate a global picture of phosphorylation events regulated by PPP1R12A in a L6 skeletal muscle cell line, which were engineered for inducible PPP1R12A knockdown. Phosphoproteomics revealed 3876 phosphorylation sites (620 were novel) in these cells. Furthermore, PPP1R12A knockdown resulted in increased overall phosphorylation in L6 cells at the basal condition, and changed phosphorylation levels for 698 sites (assigned to 295 phosphoproteins) at the basal and/or insulin-stimulated conditions. Pathway analysis on the 295 phosphoproteins revealed multiple significantly enriched pathways related to insulin signaling, such as mTOR signaling and RhoA signaling. Moreover, phosphorylation levels for numerous regulatory sites in these pathways were significantly changed due to PPP1R12A knockdown. These results indicate that PPP1R12A indeed plays a role in skeletal muscle insulin signaling, providing novel insights into the biology of insulin action. This new information may facilitate the design of experiments to better understand mechanisms underlying skeletal muscle insulin resistance and type 2 diabetes. PMID:24972320

  1. Multidimensional electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) for quantitative analysis of the proteome and phosphoproteome in clinical and biomedical research.

    PubMed

    Loroch, Stefan; Schommartz, Tim; Brune, Wolfram; Zahedi, René Peiman; Sickmann, Albert

    2015-05-01

    Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein. To address this issue, we generated a highly sensitive workflow for combined LC-MS-based quantitative proteomics and phosphoproteomics by refining an ERLIC-based 2D phosphoproteomics workflow into an ERLIC-based 3D workflow covering the global proteome as well. The resulting 3D strategy was successfully used for an in-depth quantitative analysis of both, the proteome and the phosphoproteome of murine cytomegalovirus-infected mouse fibroblasts, a model system for host cell manipulation by a virus. In a 2-plex SILAC experiment with 150 μg of a tryptic digest per condition, the 3D strategy enabled the quantification of ~75% more proteins and even ~134% more peptides compared to the 2D strategy. Additionally, we could quantify ~50% more phosphoproteins by non-phosphorylated peptides, concurrently yielding insights into changes on the levels of protein expression and phosphorylation. Beside its sensitivity, our novel three-dimensional ERLIC-strategy has the potential for semi-automated sample processing rendering it a suitable future perspective for clinical, pre-clinical and biomedical research.

  2. Quantitative Proteomic and Phosphoproteomic Analysis of H37Ra and H37Rv Strains of Mycobacterium tuberculosis.

    PubMed

    Verma, Renu; Pinto, Sneha Maria; Patil, Arun Hanumana; Advani, Jayshree; Subba, Pratigya; Kumar, Manish; Sharma, Jyoti; Dey, Gourav; Ravikumar, Raju; Buggi, Shashidhar; Satishchandra, Parthasarathy; Sharma, Kusum; Suar, Mrutyunjay; Tripathy, Srikanth Prasad; Chauhan, Devendra Singh; Gowda, Harsha; Pandey, Akhilesh; Gandotra, Sheetal; Prasad, Thottethodi Subrahmanya Keshava

    2017-03-20

    Mycobacterium tuberculosis, the causative agent of tuberculosis, accounts for 1.5 million human deaths annually worldwide. Despite efforts to eradicate tuberculosis, it still remains a deadly disease. The two best characterized strains of M. tuberculosis, virulent H37Rv and avirulent H37Ra, provide a unique platform to investigate biochemical and signaling pathways associated with pathogenicity. To delineate the biomolecular dynamics that may account for pathogenicity and attenuation of virulence in M. tuberculosis, we compared the proteome and phosphoproteome profiles of H37Rv and H37Ra strains. Quantitative phosphoproteomic analysis was performed using high-resolution Fourier transform mass spectrometry. Analysis of exponential and stationary phases of these strains resulted in identification and quantitation of 2709 proteins along with 512 phosphorylation sites derived from 257 proteins. In addition to confirming the presence of previously described M. tuberculosis phosphorylated proteins, we identified 265 novel phosphorylation sites. Quantitative proteomic analysis revealed more than five-fold upregulation of proteins belonging to virulence associated type VII bacterial secretion system in H37Rv when compared to those in H37Ra. We also identified 84 proteins, which exhibited changes in phosphorylation levels between the virulent and avirulent strains. Bioinformatics analysis of the proteins altered in their level of expression or phosphorylation revealed enrichment of pathways involved in fatty acid biosynthesis and two-component regulatory system. Our data provides a resource for further exploration of functional differences at molecular level between H37Rv and H37Ra, which will ultimately explain the molecular underpinnings that determine virulence in tuberculosis.

  3. Quantitative Phosphoproteomic Analysis of Soybean Root Hairs Inoculated with Bradyrhizobium japonicum

    SciTech Connect

    Nguyen, Tran H.; Brechenmacher, Laurent; Aldrich, Joshua T.; Clauss, Therese RW; Gritsenko, Marina A.; Hixson, Kim K.; Libault, Marc; Tanaka, Kiwamu; Yang, Feng; Yao, Qiuming; Pasa-Tolic, Ljiljana; Xu, Dong; Nguyen, Henry T.; Stacey, Gary

    2012-11-11

    Root hairs are single hair-forming cells on roots that function to increase root surface area, enhancing water and nutrient uptake. In leguminous plants, root hairs also play a critical role as the site of infection by symbiotic nitrogen fixing rhizobia, leading to the formation of a novel organ, the nodule. The initial steps in the rhizobia-root hair infection process are known to involve specific receptor kinases and subsequent kinase cascades. Here, we characterize the phosphoproteome of the root hairs and the corresponding stripped roots (i.e., roots from which root hairs were removed) during rhizobial colonization and infection to gain insight into the molecular mechanism of root hair cell biology. We chose soybean (Glycine max L.), one of the most important crop plants in the legume family, for this study because of its larger root size, which permits isolation of sufficient root hair material for phosphoproteomic analysis. Phosphopeptides derived from root hairs and stripped roots, mock inoculated or inoculated with the soybean-specific rhizobium Bradyrhizobium japonicum, were labeled with the isobaric tag 8-plex ITRAQ, enriched using Ni-NTA magnetic beads and subjected to nRPLC-MS/MS analysis using HCD and decision tree guided CID/ETD strategy. A total of 1,625 unique phosphopeptides, spanning 1,659 non-redundant phosphorylation sites, were detected from 1,126 soybean phosphoproteins. Among them, 273 phosphopeptides corresponding to 240 phosphoproteins were found to be significantly regulated (>1.5 fold abundance change) in response to inoculation with B. japonicum. The data reveal unique features of the soybean root hair phosphoproteome, including root hair and stripped root-specific phosphorylation suggesting a complex network of kinase-substrate and phosphatase-substrate interactions in response to rhizobial inoculation.

  4. Discovery of mouse spleen signaling responses to anthrax using label-free quantitative phosphoproteomics via mass spectrometry.

    PubMed

    Manes, Nathan P; Dong, Li; Zhou, Weidong; Du, Xiuxia; Reghu, Nikitha; Kool, Arjan C; Choi, Dahan; Bailey, Charles L; Petricoin, Emanuel F; Liotta, Lance A; Popov, Serguei G

    2011-03-01

    Inhalational anthrax is caused by spores of the bacterium Bacillus anthracis (B. anthracis), and is an extremely dangerous disease that can kill unvaccinated victims within 2 weeks. Modern antibiotic-based therapy can increase the survival rate to ∼50%, but only if administered presymptomatically (within 24-48 h of exposure). To discover host signaling responses to presymptomatic anthrax, label-free quantitative phosphoproteomics via liquid chromatography coupled to mass spectrometry was used to compare spleens from uninfected and spore-challenged mice over a 72 h time-course. Spleen proteins were denatured using urea, reduced using dithiothreitol, alkylated using iodoacetamide, and digested into peptides using trypsin, and the resulting phosphopeptides were enriched using titanium dioxide solid-phase extraction and analyzed by nano-liquid chromatography-Linear Trap Quadrupole-Orbitrap-MS(/MS). The fragment ion spectra were processed using DeconMSn and searched using both Mascot and SEQUEST resulting in 252,626 confident identifications of 6248 phosphopeptides (corresponding to 5782 phosphorylation sites). The precursor ion spectra were deisotoped using Decon2LS and aligned using MultiAlign resulting in the confident quantitation of 3265 of the identified phosphopeptides. ANOVAs were used to produce a q-value ranked list of host signaling responses. Late-stage (48-72 h postchallenge) Sterne strain (lethal) infections resulted in global alterations to the spleen phosphoproteome. In contrast, ΔSterne strain (asymptomatic; missing the anthrax toxin) infections resulted in 188 (5.8%) significantly altered (q<0.05) phosphopeptides. Twenty-six highly tentative phosphorylation responses to early-stage (24 h postchallenge) anthrax were discovered (q<0.5), and ten of these originated from eight proteins that have known roles in the host immune response. These tentative early-anthrax host response signaling events within mouse spleens may translate into presymptomatic

  5. Discovery of Mouse Spleen Signaling Responses to Anthrax using Label-Free Quantitative Phosphoproteomics via Mass Spectrometry*

    PubMed Central

    Manes, Nathan P.; Dong, Li; Zhou, Weidong; Du, Xiuxia; Reghu, Nikitha; Kool, Arjan C.; Choi, Dahan; Bailey, Charles L.; Petricoin, Emanuel F.; Liotta, Lance A.; Popov, Serguei G.

    2011-01-01

    Inhalational anthrax is caused by spores of the bacterium Bacillus anthracis (B. anthracis), and is an extremely dangerous disease that can kill unvaccinated victims within 2 weeks. Modern antibiotic-based therapy can increase the survival rate to ∼50%, but only if administered presymptomatically (within 24–48 h of exposure). To discover host signaling responses to presymptomatic anthrax, label-free quantitative phosphoproteomics via liquid chromatography coupled to mass spectrometry was used to compare spleens from uninfected and spore-challenged mice over a 72 h time-course. Spleen proteins were denatured using urea, reduced using dithiothreitol, alkylated using iodoacetamide, and digested into peptides using trypsin, and the resulting phosphopeptides were enriched using titanium dioxide solid-phase extraction and analyzed by nano-liquid chromatography-Linear Trap Quadrupole-Orbitrap-MS(/MS). The fragment ion spectra were processed using DeconMSn and searched using both Mascot and SEQUEST resulting in 252,626 confident identifications of 6248 phosphopeptides (corresponding to 5782 phosphorylation sites). The precursor ion spectra were deisotoped using Decon2LS and aligned using MultiAlign resulting in the confident quantitation of 3265 of the identified phosphopeptides. ANOVAs were used to produce a q-value ranked list of host signaling responses. Late-stage (48–72 h postchallenge) Sterne strain (lethal) infections resulted in global alterations to the spleen phosphoproteome. In contrast, ΔSterne strain (asymptomatic; missing the anthrax toxin) infections resulted in 188 (5.8%) significantly altered (q<0.05) phosphopeptides. Twenty-six highly tentative phosphorylation responses to early-stage (24 h postchallenge) anthrax were discovered (q<0.5), and ten of these originated from eight proteins that have known roles in the host immune response. These tentative early-anthrax host response signaling events within mouse spleens may translate into presymptomatic

  6. Quantitative analysis of changes in the phosphoproteome of maize induced by the plant hormone salicylic acid

    PubMed Central

    Wu, Liuji; Hu, Xiuli; Wang, Shunxi; Tian, Lei; Pang, Yanjie; Han, Zanping; Wu, Liancheng; Chen, Yanhui

    2015-01-01

    Phytohormone salicylic acid (SA) plays an important role in regulating various physiological and biochemical processes. Our previous study identified several protein kinases responsive to SA, suggesting that phosphorylation events play an important role in the plant response to SA. In this study, we characterized the phosphoproteome of maize in response to SA using isotope tags for relative and absolute quantification (iTRAQ) technology and TiO2 enrichment method. Based on LC-MS/MS analysis, we found a total of 858 phosphoproteins among 1495 phosphopeptides. Among them, 291 phosphopeptides corresponding to 244 phosphoproteins were found to be significantly changed after SA treatment. The phosphoproteins identified are involved in a wide range of biological processes, which indicate that the response to SA encompasses a reformatting of major cellular processes. Furthermore, some of the phosphoproteins which were not previously known to be involved with SA were found to have significantly changed phosphorylation levels. Many of these changes are phosphorylation decreases, indicating that other currently unknown SA signaling pathways that result in decreased phosphorylation of downstream targets must be involved. Our study represents the first attempt at global phosphoproteome profiling in response to SA, and provides a better understanding of the molecular mechanisms regulated by SA. PMID:26659305

  7. Quantitative Phosphoproteomics Reveals Signaling Mechanisms Associated with Rapid Cold Hardening in a Chill-Tolerant Fly.

    PubMed

    Teets, Nicholas M; Denlinger, David L

    2016-08-05

    Rapid cold hardening (RCH) is a physiological adaptation in which brief chilling (minutes to hours) significantly enhances the cold tolerance of insects. RCH allows insects to cope with sudden cold snaps and diurnal variation in temperature, but the mechanistic basis of this rapid stress response is poorly understood. Here, we used phosphoproteomics to identify phosphorylation-mediated signaling events that are regulated by chilling that induces RCH. Phosphoproteomic changes were measured in both brain and fat bodies, two tissues that are essential for sensing cold and coordinating RCH at the organismal level. Tissues were chilled ex vivo, and changes in phosphoprotein abundance were measured using 2D electrophoresis coupled with Pro-Q diamond labeling of phosphoproteins followed by protein identification via LC-MS/MS. In both tissues, we observed an abundance of protein phosphorylation events in response to chilling. Some of the proteins regulated by RCH-inducing chilling include proteins involved in cytoskeletal reorganization, heat shock proteins, and proteins involved in the degradation of damaged cellular components via the proteasome and autophagosome. Our results suggest that phosphorylation-mediated signaling cascades are major drivers of RCH and enhance our mechanistic understanding of this complex phenotype.

  8. Quantitative analysis of changes in the phosphoproteome of maize induced by the plant hormone salicylic acid.

    PubMed

    Wu, Liuji; Hu, Xiuli; Wang, Shunxi; Tian, Lei; Pang, Yanjie; Han, Zanping; Wu, Liancheng; Chen, Yanhui

    2015-12-11

    Phytohormone salicylic acid (SA) plays an important role in regulating various physiological and biochemical processes. Our previous study identified several protein kinases responsive to SA, suggesting that phosphorylation events play an important role in the plant response to SA. In this study, we characterized the phosphoproteome of maize in response to SA using isotope tags for relative and absolute quantification (iTRAQ) technology and TiO2 enrichment method. Based on LC-MS/MS analysis, we found a total of 858 phosphoproteins among 1495 phosphopeptides. Among them, 291 phosphopeptides corresponding to 244 phosphoproteins were found to be significantly changed after SA treatment. The phosphoproteins identified are involved in a wide range of biological processes, which indicate that the response to SA encompasses a reformatting of major cellular processes. Furthermore, some of the phosphoproteins which were not previously known to be involved with SA were found to have significantly changed phosphorylation levels. Many of these changes are phosphorylation decreases, indicating that other currently unknown SA signaling pathways that result in decreased phosphorylation of downstream targets must be involved. Our study represents the first attempt at global phosphoproteome profiling in response to SA, and provides a better understanding of the molecular mechanisms regulated by SA.

  9. Identification of Putative Mek1 Substrates during Meiosis in Saccharomyces cerevisiae Using Quantitative Phosphoproteomics

    PubMed Central

    Suhandynata, Raymond T.; Wan, Lihong; Zhou, Huilin; Hollingsworth, Nancy M.

    2016-01-01

    Meiotic recombination plays a key role in sexual reproduction as it generates crossovers that, in combination with sister chromatid cohesion, physically connect homologous chromosomes, thereby promoting their proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs) catalyzed by the evolutionarily conserved, topoisomerase-like protein Spo11. Repair of these DSBs is highly regulated to create crossovers between homologs that are distributed throughout the genome. This repair requires the presence of the mitotic recombinase, Rad51, as well as the strand exchange activity of the meiosis-specific recombinase, Dmc1. A key regulator of meiotic DSB repair in Saccharomyces cerevisiae is the meiosis-specific kinase Mek1, which promotes interhomolog strand invasion and is required for the meiotic recombination checkpoint and the crossover/noncrossover decision. Understanding how Mek1 regulates meiotic recombination requires the identification of its substrates. Towards that end, an unbiased phosphoproteomic approach utilizing Stable Isotope Labeling by Amino Acids in Cells (SILAC) was utilized to generate a list of potential Mek1 substrates, as well as proteins containing consensus phosphorylation sites for cyclin-dependent kinase, the checkpoint kinases, Mec1/Tel1, and the polo-like kinase, Cdc5. These experiments represent the first global phosphoproteomic dataset for proteins in meiotic budding yeast. PMID:27214570

  10. Quantitative phosphoproteomic analyses of the inferior parietal lobule from three different pathological stages of Alzheimer's disease.

    PubMed

    Triplett, Judy C; Swomley, Aaron M; Cai, Jian; Klein, Jon B; Butterfield, D Allan

    2016-01-01

    Alzheimer's disease (AD), the most common age-related neurodegenerative disorder, is clinically characterized by progressive neuronal loss resulting in loss of memory and dementia. AD is histopathologically characterized by the extensive distribution of senile plaques and neurofibrillary tangles, and synapse loss. Amnestic mild cognitive impairment (MCI) is generally accepted to be an early stage of AD. MCI subjects have pathology and symptoms that fall on the scale intermediately between 'normal' cognition with little or no pathology and AD. A rare number of individuals, who exhibit normal cognition on psychometric tests but whose brains show widespread postmortem AD pathology, are classified as 'asymptomatic' or 'preclinical' AD (PCAD). In this study, we evaluated changes in protein phosphorylation states in the inferior parietal lobule of subjects with AD, MCI, PCAD, and control brain using a 2-D PAGE proteomics approach in conjunction with Pro-Q Diamond phosphoprotein staining. Statistically significant changes in phosphorylation levels were found in 19 proteins involved in energy metabolism, neuronal plasticity, signal transduction, and oxidative stress response. Changes in the disease state phosphoproteome may provide insights into underlying mechanisms for the preservation of memory with expansive AD pathology in PCAD and the progressive memory loss in amnestic MCI that escalates to the dementia and the characteristic pathology of AD brain.

  11. Quantitative phosphoproteomic analysis reveals system-wide signaling pathways regulated by site-specific phosphorylation on Keratin-8 in skin squamous cell carcinoma derived cell- line.

    PubMed

    Tiwari, Richa; Sahu, Indrajit; Soni, Bihari Lal; Sathe, Gajanan J; Datta, Keshava K; Thapa, Pankaj; Sinha, Shruti; Vadivel, Chella Krishna; Dhaka, Bharti; Gowda, Harsha; Vaidya, Milind M

    2017-02-07

    Keratin 8/18, a simple epithelia specific keratin pair, is often aberrantly expressed in squamous cell carcinomas (SCC) where its expression is correlated with increased invasion and poor prognosis. Majority of Keratin 8 (K8) functions are governed by its phosphorylation at Serine(73) (head-domain) and Serine(431) (tail-domain) residues. Although, deregulation of K8 phosphorylation is associated with progression of different carcinomas, its role in skin-SCC and the underlying mechanism is obscure. In this direction, we performed TMT-based quantitative phosphoproteomics by expressing K8 wild type, phosphodead and phosphomimetic mutants in K8-deficient A431 cells. Further analysis of our phosphoproteomics data showed a significant proportion of total phosphoproteome associated with migratory, proliferative and invasive potential of these cells to be differentially phosphorylated. Differential phosphorylation of CDK1(T14,Y15) , EIF4EBP1(T46,T50) , EIF4B(S422) , AKT1S1T246,S247, CTTN1(T401,S405,) Y421 & CAP1(S307/309) in K8-S73A/D mutant and CTTN1(T401,S405,Y421) , BUB1B(S1043) & CARHSP1(S30,S32) in K8-S431A/D mutants as well as some anonymous phosphosites including MYC(S176) , ZYX(S344) and PNN(S692) could be potential candidates associated with K8 phosphorylation mediated tumorigenicity. Biochemical validation followed by phenotypic analysis further confirmed our quantitative phosphoproteomics data. In conclusion, our study provides the first global picture of K8 site- specific phosphorylation function in neoplastic progression of A431 cells and suggests various potential starting points for further mechanistic studies. This article is protected by copyright. All rights reserved.

  12. Quantitative phosphoproteomics revealed interplay between Syk and Lyn in the resistance to nilotinib in chronic myeloid leukemia cells.

    PubMed

    Gioia, Romain; Leroy, Cédric; Drullion, Claire; Lagarde, Valérie; Etienne, Gabriel; Dulucq, Stéphanie; Lippert, Eric; Roche, Serge; Mahon, François-Xavier; Pasquet, Jean-Max

    2011-08-25

    In this study, we have addressed how Lyn kinase signaling mediates nilotinib-resistance by quantitative phospho-proteomics using Stable Isotope Labeling with Amino acid in Cell culture. We have found an increased tyrosine phosphorylation of 2 additional tyrosine kinases in nilotinib-resistant cells: the spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl. This increased tyrosine phosphorylation involved an interaction of these tyrosine kinases with Lyn. Inhibition of Syk by the inhibitors R406 or BAY 61-3606 or by RNA interference restored the capacity of nilotinib to inhibit cell proliferation. Conversely, coexpression of Lyn and Syk were required to fully induce resistance to nilotinib in drug-sensitive cells. Surprisingly, the knockdown of Syk also strongly decreased tyrosine phosphorylation of Lyn and Axl, thus uncovering interplay between Syk and Lyn. We have shown the involvement of the adaptor protein CDCP-1 in resistance to nilotinib. Interestingly, the expression of Axl and CDCP1 were found increased both in a nilotinib-resistant cell line and in nilotinib-resistant CML patients. We conclude that an oncogenic signaling mediated by Lyn and Syk can bypass the need of Bcr-Abl in CML cells. Thus, targeting these kinases may be of therapeutic value to override imatinib or nilotinib resistance in CML.

  13. Quantitative phosphoproteomics of murine Fmr1-KO cell lines provides new insights into FMRP-dependent signal transduction mechanisms.

    PubMed

    Matic, Katarina; Eninger, Timo; Bardoni, Barbara; Davidovic, Laetitia; Macek, Boris

    2014-10-03

    Fragile X mental retardation protein (FMRP) is an RNA-binding protein that has a major effect on neuronal protein synthesis. Transcriptional silencing of the FMR1 gene leads to loss of FMRP and development of Fragile X syndrome (FXS), the most common known hereditary cause of intellectual impairment and autism. Here we utilize SILAC-based quantitative phosphoproteomics to analyze murine FMR1(-) and FMR1(+) fibroblastic cell lines derived from FMR1-KO embryos to identify proteins and phosphorylation sites dysregulated as a consequence of FMRP loss. We quantify FMRP-related changes in the levels of 5,023 proteins and 6,133 phosphorylation events and map them onto major signal transduction pathways. Our study confirms global downregulation of the MAPK/ERK pathway and decrease in phosphorylation level of ERK1/2 in the absence of FMRP, which is connected to attenuation of long-term potentiation. We detect differential expression of several key proteins from the p53 pathway, pointing to the involvement of p53 signaling in dysregulated cell cycle control in FXS. Finally, we detect differential expression and phosphorylation of proteins involved in pre-mRNA processing and nuclear transport, as well as Wnt and calcium signaling, such as PLC, PKC, NFAT, and cPLA2. We postulate that calcium homeostasis is likely affected in molecular pathogenesis of FXS.

  14. Quantitative Circadian Phosphoproteomic Analysis of Arabidopsis Reveals Extensive Clock Control of Key Components in Physiological, Metabolic, and Signaling Pathways.

    PubMed

    Choudhary, Mani Kant; Nomura, Yuko; Wang, Lei; Nakagami, Hirofumi; Somers, David E

    2015-08-01

    The circadian clock provides adaptive advantages to an organism, resulting in increased fitness and survival. The phosphorylation events that regulate circadian-dependent signaling and the processes which post-translationally respond to clock-gated signals are largely unknown. To better elucidate post-translational events tied to the circadian system we carried out a survey of circadian-regulated protein phosphorylation events in Arabidopsis seedlings. A large-scale mass spectrometry-based quantitative phosphoproteomics approach employing TiO2-based phosphopeptide enrichment techniques identified and quantified 1586 phosphopeptides on 1080 protein groups. A total of 102 phosphopeptides displayed significant changes in abundance, enabling the identification of specific patterns of response to circadian rhythms. Our approach was sensitive enough to quantitate oscillations in the phosphorylation of low abundance clock proteins (early flowering4; ELF4 and pseudoresponse regulator3; PRR3) as well as other transcription factors and kinases. During constant light, extensive cyclic changes in phosphorylation status occurred in critical regulators, implicating direct or indirect regulation by the circadian system. These included proteins influencing transcriptional regulation, translation, metabolism, stress and phytohormones-mediated responses. We validated our analysis using the elf4-211 allele, in which an S45L transition removes the phosphorylation herein identified. We show that removal of this phosphorylatable site diminishes interaction with early flowering3 (ELF3), a key partner in a tripartite evening complex required for circadian cycling. elf4-211 lengthens period, which increases with increasing temperature, relative to the wild type, resulting in a more stable temperature compensation of circadian period over a wider temperature range.

  15. Phosphoproteomics in Cancer

    PubMed Central

    Harsha, H. C.; Pandey, Akhilesh

    2010-01-01

    Reversible protein phosphorylation serves as a basis for regulating a number of cellular processes. Aberrant activation of kinase signaling pathways is commonly associated with several cancers. Recent developments in phosphoprotein/phosphopeptide enrichment strategies and quantitative mass spectrometry have resulted in robust pipelines for high-throughput characterization of phosphorylation in a global fashion. Today, it is possible to profile site-specific phosphorylation events on thousands of proteins in a single experiment. The potential of this approach is already being realized to characterize signaling pathways that govern oncogenesis. In addition, chemical proteomic strategies have been used to unravel targets of kinase inhibitors, which are otherwise difficult to characterize. This review summarizes various approaches used for analysis of the phosphoproteome in general, and protein kinases in particular, highlighting key cancer phosphoproteomic studies. PMID:20937571

  16. Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action

    PubMed Central

    Wang, Pengcheng; Xue, Liang; Batelli, Giorgia; Lee, Shinyoung; Hou, Yueh-Ju; Van Oosten, Michael J.; Zhang, Huiming; Tao, W. Andy; Zhu, Jian-Kang

    2013-01-01

    Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments. PMID:23776212

  17. Quantitative Phosphoproteome Analysis of Lysophosphatidic Acid Induced Chemotaxis applying Dual-step ¹⁸O Labeling Coupled with Immobilized Metal-ion Affinity Chromatography

    SciTech Connect

    Ding, Shi-Jian; Wang, Yingchun; Jacobs, Jon M.; Qian, Weijun; Yang, Feng; Tolmachev, Aleksey V.; Du, Xiuxia; Wang, Wei; Moore, Ronald J.; Monroe, Matthew E.; Purvine, Samuel O.; Waters, Katrina M.; Heibeck, Tyler H.; Adkins, Joshua N.; Camp, David G.; Klemke, Richard L.; Smith, Richard D.

    2008-10-01

    Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in a variety of different cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its applications for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed 16O/18O labeling plus 16O/18O-methanol esterification labeling for quantitation, a macro- Immobilized Metal-ion Affinity Chromatography trap for phosphopeptide enrichment, and a monolithic capillary column with integrated electrospray emitter. LC separation and MS/MS is followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer and complementary searching algorithms for interpreting MS/MS spectra. Protein phosphorylation involved in various signaling pathways of cell migration were identified and quantified, such as mitogen-activated protein kinase 1, dual-specificity mitogen-activated protein kinase kinase 2, and dual-specificity tyrosine-phosphorylation regulated kinase 1b, and a number of Rho GTPase-activating proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with gradient sensing and cell chemotaxis.

  18. Quantitative Phosphoproteomic Analysis Reveals a Role for Serine and Threonine Kinases in the Cytoskeletal Reorganization in Early T Cell Receptor Activation in Human Primary T Cells*

    PubMed Central

    Ruperez, Patricia; Gago-Martinez, Ana; Burlingame, A. L.; Oses-Prieto, Juan A.

    2012-01-01

    Protein phosphorylation-dephosphorylation events play a primary role in regulation of almost all aspects of cell function including signal transduction, cell cycle, or apoptosis. Thus far, T cell phosphoproteomics have focused on analysis of phosphotyrosine residues, and little is known about the role of serine/threonine phosphorylation in early activation of the T cell receptor (TCR). Therefore, we performed a quantitative mass spectrometry-based analysis of the global phosphoproteome of human primary T cells in response to 5 min of TCR activation with anti-CD3 antibody. Combining immunoprecipitation with an antiphosphotyrosine antibody, titanium dioxide phosphopeptide enrichment, isobaric tag for the relative and absolute quantitation methodology, and strong cation exchange separation, we were able to identify 2814 phosphopeptides. These unique sites were employed to investigate the site-specific phosphorylation dynamics. Five hundred and seventeen phosphorylation sites showed TCR-responsive changes. We found that upon 5 min of stimulation of the TCR, specific serine and threonine kinase motifs are overrepresented in the set of responsive phosphorylation sites. These phosphorylation events targeted proteins with many different activities and are present in different subcellular locations. Many of these proteins are involved in intracellular signaling cascades related mainly to cytoskeletal reorganization and regulation of small GTPase-mediated signal transduction, probably involved in the formation of the immune synapse. PMID:22499768

  19. Targeted quantitative phosphoproteomic analysis of erythrocyte membranes during blood bank storage.

    PubMed

    Rinalducci, Sara; Longo, Valentina; Ceci, Luigi R; Zolla, Lello

    2015-02-01

    One of the hallmarks of blood bank stored red blood cells (RBCs) is the irreversible transition from a discoid to a spherocyte-like morphology with membrane perturbation and cytoskeleton disorders. Therefore, identification of the storage-associated modifications in the protein-protein interactions between the cytoskeleton and the lipid bilayer may contribute to enlighten the molecular mechanisms involved in the alterations of mechanical properties of stored RBCs. Here we report the results obtained analyzing RBCs after 0, 21 and 35 days of storage under standard blood banking conditions by label free mass spectrometry (MS)-based experiments. We could quantitatively measure changes in the phosphorylation level of crucial phosphopeptides belonging to β-spectrin, ankyrin-1, α-adducin, dematin, glycophorin A and glycophorin C proteins. Data have been validated by both western blotting and pseudo-Multiple Reaction Monitoring (MRM). Although each phosphopeptide showed a distinctive trend, a sharp increase in the phosphorylation level during the storage duration was observed. Phosphopeptide mapping and structural modeling analysis indicated that the phosphorylated residues localize in protein functional domains fundamental for the maintenance of membrane structural integrity. Along with previous morphological evidence acquired by electron microscopy, our results seem to indicate that 21-day storage may represent a key point for the molecular processes leading to the erythrocyte deformability reduction observed during blood storage. These findings could therefore be helpful in understanding and preventing the morphology-linked mechanisms responsible for the post-transfusion survival of preserved RBCs.

  20. Quantitative Site-Specific Phosphoproteomics of Trichoderma reesei Signaling Pathways upon Induction of Hydrolytic Enzyme Production.

    PubMed

    Nguyen, Elizabeth V; Imanishi, Susumu Y; Haapaniemi, Pekka; Yadav, Avinash; Saloheimo, Markku; Corthals, Garry L; Pakula, Tiina M

    2016-02-05

    The filamentous fungus Trichoderma reesei is used for industrial production of secreted enzymes including carbohydrate active enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by T. reesei is influenced by the carbon source it grows on, where the regulation system controlling hydrolase genes involves various signaling pathways. T. reesei was cultivated in the presence of sorbitol, a carbon source that does not induce the production of cellulases and hemicellulases, and then exposed to either sophorose or spent-grain extract, which are efficient inducers of the enzyme production. Specific changes at phosphorylation sites were investigated in relation to the production of cellulases and hemicellulases using an MS-based framework. Proteome-wide phosphorylation following carbon source exchange was investigated in the early stages of induction: 0, 2, 5, and 10 min. The workflow involved sequential trypsin digestion, TiO2 enrichment, and MS analysis using a Q Exactive mass spectrometer. We report on the identification and quantitation of 1721 phosphorylation sites. Investigation of the data revealed a complex signaling network activated upon induction involving components related to light-mediated cellulase induction, osmoregulation, and carbon sensing. Changes in protein phosphorylation were detected in the glycolytic pathway, suggesting an inhibition of glucose catabolism at 10 min after the addition of sophorose and as early as 2 min after the addition of spent-grain extract. Differential phosphorylation of factors related to carbon storage, intracellular trafficking, cytoskeleton, and cellulase gene regulation were also observed.

  1. iTRAQ labeling is superior to mTRAQ for quantitative global proteomics and phosphoproteomics.

    PubMed

    Mertins, Philipp; Udeshi, Namrata D; Clauser, Karl R; Mani, D R; Patel, Jinal; Ong, Shao-en; Jaffe, Jacob D; Carr, Steven A

    2012-06-01

    Labeling of primary amines on peptides with reagents containing stable isotopes is a commonly used technique in quantitative mass spectrometry. Isobaric labeling techniques such as iTRAQ™ or TMT™ allow for relative quantification of peptides based on ratios of reporter ions in the low m/z region of spectra produced by precursor ion fragmentation. In contrast, nonisobaric labeling with mTRAQ™ yields precursors with different masses that can be directly quantified in MS1 spectra. In this study, we compare iTRAQ- and mTRAQ-based quantification of peptides and phosphopeptides derived from EGF-stimulated HeLa cells. Both labels have identical chemical structures, therefore precursor ion- and fragment ion-based quantification can be directly compared. Our results indicate that iTRAQ labeling has an additive effect on precursor intensities, whereas mTRAQ labeling leads to more redundant MS2 scanning events caused by triggering on the same peptide with different mTRAQ labels. We found that iTRAQ labeling quantified nearly threefold more phosphopeptides (12,129 versus 4,448) and nearly twofold more proteins (2,699 versus 1,597) than mTRAQ labeling. Although most key proteins in the EGFR signaling network were quantified with both techniques, iTRAQ labeling allowed quantification of twice as many kinases. Accuracy of reporter ion quantification by iTRAQ is adversely affected by peptides that are cofragmented in the same precursor isolation window, dampening observed ratios toward unity. However, because of tighter overall iTRAQ ratio distributions, the percentage of statistically significantly regulated phosphopeptides and proteins detected by iTRAQ and mTRAQ was similar. We observed a linear correlation of logarithmic iTRAQ to mTRAQ ratios over two orders of magnitude, indicating a possibility to correct iTRAQ ratios by an average compression factor. Spike-in experiments using peptides of defined ratios in a background of nonregulated peptides show that i

  2. Quantitative Measurement of Phosphoproteome Response to Osmotic Stress in Arabidopsis Based on Library-Assisted eXtracted Ion Chromatogram (LAXIC)*

    PubMed Central

    Xue, Liang; Wang, Pengcheng; Wang, Lianshui; Renzi, Emily; Radivojac, Predrag; Tang, Haixu; Arnold, Randy; Zhu, Jian-Kang; Tao, W. Andy

    2013-01-01

    Global phosphorylation changes in plants in response to environmental stress have been relatively poorly characterized to date. Here we introduce a novel mass spectrometry-based label-free quantitation method that facilitates systematic profiling plant phosphoproteome changes with high efficiency and accuracy. This method employs synthetic peptide libraries tailored specifically as internal standards for complex phosphopeptide samples and accordingly, a local normalization algorithm, LAXIC, which calculates phosphopeptide abundance normalized locally with co-eluting library peptides. Normalization was achieved in a small time frame centered to each phosphopeptide to compensate for the diverse ion suppression effect across retention time. The label-free LAXIC method was further treated with a linear regression function to accurately measure phosphoproteome responses to osmotic stress in Arabidopsis. Among 2027 unique phosphopeptides identified and 1850 quantified phosphopeptides in Arabidopsis samples, 468 regulated phosphopeptides representing 497 phosphosites have shown significant changes. Several known and novel components in the abiotic stress pathway were identified, illustrating the capability of this method to identify critical signaling events among dynamic and complex phosphorylation. Further assessment of those regulated proteins may help shed light on phosphorylation response to osmotic stress in plants. PMID:23660473

  3. Quantitative label-free phosphoproteomics of six different life stages of the late blight pathogen Phytophthora infestans reveals abundant phosphorylation of members of the CRN effector family.

    PubMed

    Resjö, Svante; Ali, Ashfaq; Meijer, Harold J G; Seidl, Michael F; Snel, Berend; Sandin, Marianne; Levander, Fredrik; Govers, Francine; Andreasson, Erik

    2014-04-04

    The oomycete Phytophthora infestans is the causal agent of late blight in potato and tomato. Since the underlying processes that govern pathogenicity and development in P. infestans are largely unknown, we have performed a large-scale phosphoproteomics study of six different P. infestans life stages. We have obtained quantitative data for 2922 phosphopeptides and compared their abundance. Life-stage-specific phosphopeptides include ATP-binding cassette transporters and a kinase that only occurs in appressoria. In an extended data set, we identified 2179 phosphorylation sites and deduced 22 phosphomotifs. Several of the phosphomotifs matched consensus sequences of kinases that occur in P. infestans but not Arabidopsis. In addition, we detected tyrosine phosphopeptides that are potential targets of kinases resembling mammalian tyrosine kinases. Among the phosphorylated proteins are members of the RXLR and Crinkler effector families. The latter are phosphorylated in several life stages and at multiple positions, in sites that are conserved between different members of the Crinkler family. This indicates that proteins in the Crinkler family have functions beyond their putative role as (necrosis-inducing) effectors. This phosphoproteomics data will be instrumental for studies on oomycetes and host-oomycete interactions. The data sets have been deposited to ProteomeXchange (identifier PXD000433).

  4. Phosphoproteomics and molecular cardiology: techniques, applications and challenges.

    PubMed

    Sun, Zeyu; Hamilton, Karyn L; Reardon, Kenneth F

    2012-09-01

    Protein phosphorylation has been widely documented as a key regulatory and signaling mechanism associated with many cardiac diseases. Recent advances in phosphoproteomic technologies such as phosphopeptide enrichment, novel mass spectrometry applications, and bioinformatic tools have resulted in high-throughput identification and quantitation of protein phosphorylation in a global manner. This review summarizes mainstream phosphoproteomic workflows and highlights the most recent applications of phosphoproteomics used in a range of molecular cardiology research.

  5. Quantitative Phosphoproteome Analysis of Bacillus subtilis Reveals Novel Substrates of the Kinase PrkC and Phosphatase PrpC*

    PubMed Central

    Ravikumar, Vaishnavi; Shi, Lei; Krug, Karsten; Derouiche, Abderahmane; Jers, Carsten; Cousin, Charlotte; Kobir, Ahasanul; Mijakovic, Ivan; Macek, Boris

    2014-01-01

    Reversible protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) residues plays a critical role in regulation of vital processes in the cell. Despite of considerable progress in our understanding of the role of this modification in bacterial physiology, the dynamics of protein phosphorylation during bacterial growth has rarely been systematically addressed. In addition, little is known about in vivo substrates of bacterial Ser/Thr/Tyr kinases and phosphatases. An excellent candidate to study these questions is the Gram-positive bacterium Bacillus subtilis, one of the most intensively investigated bacterial model organism with both research and industrial applications. Here we employed gel-free phosphoproteomics combined with SILAC labeling and high resolution mass spectrometry to study the proteome and phosphoproteome dynamics during the batch growth of B. subtilis. We measured the dynamics of 1666 proteins and 64 phosphorylation sites in five distinct phases of growth. Enzymes of the central carbon metabolism and components of the translation machinery appear to be highly phosphorylated in the stationary phase, coinciding with stronger expression of Ser/Thr kinases. We further used the SILAC workflow to identify novel putative substrates of the Ser/Thr kinase PrkC and the phosphatase PrpC during stationary phase. The overall number of putative substrates was low, pointing to a high kinase and phosphatase specificity. One of the phosphorylation sites affected by both, PrkC and PrpC, was the Ser281 on the oxidoreductase YkwC. We showed that PrkC phosphorylates and PrpC dephosphorylates YkwC in vitro and that phosphorylation at Ser281 abolishes the oxidoreductase activity of YkwC in vitro and in vivo. Our results present the most detailed phosphoproteomic analysis of B. subtilis growth to date and provide the first global in vivo screen of PrkC and PrpC substrates. PMID:24390483

  6. Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling

    PubMed Central

    Kauko, Otto; Laajala, Teemu Daniel; Jumppanen, Mikael; Hintsanen, Petteri; Suni, Veronika; Haapaniemi, Pekka; Corthals, Garry; Aittokallio, Tero; Westermarck, Jukka; Imanishi, Susumu Y.

    2015-01-01

    Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantification. To allow data normalization in situations where depletion of RAS or PP2A inhibitor CIP2A causes a large uni-directional change in the phosphopeptide abundance, we developed a novel normalization strategy, named pairwise normalization. This normalization is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of the pairwise normalization was verified by various independent methods. Additionally, we demonstrate how the selected normalization method influences the downstream analyses and interpretation of pathway activities. Consequently, bioinformatics analysis of RAS and CIP2A regulated phosphoproteomes revealed a significant overlap in their functional pathways. This is most likely biologically meaningful as we observed a synergistic survival effect between CIP2A and RAS expression as well as KRAS activating mutations in TCGA pan-cancer data set, and synergistic relationship between CIP2A and KRAS depletion in colony growth assays. PMID:26278961

  7. Quantitative expression proteomics and phosphoproteomics profile of brain from PINK1 knockout mice: insights into mechanisms of familial Parkinson's disease.

    PubMed

    Triplett, Judy C; Zhang, Zhaoshu; Sultana, Rukhsana; Cai, Jian; Klein, Jon B; Büeler, Hansruedi; Butterfield, David Allan

    2015-06-01

    Parkinson's disease (PD) is an age-related, neurodegenerative motor disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of α-synuclein-containing protein aggregates. Mutations in the mitochondrial Ser/Thr kinase PTEN-induced kinase 1 (PINK1) are associated with an autosomal recessive familial form of early-onset PD. Recent studies have suggested that PINK1 plays important neuroprotective roles against mitochondrial dysfunction by phosphorylating and recruiting Parkin, a cytosolic E3 ubiquitin ligase, to facilitate elimination of damaged mitochondria via autophagy-lysosomal pathways. Loss of PINK1 in cells and animals leads to various mitochondrial impairments and oxidative stress, culminating in dopaminergic neuronal death in humans. Using a 2-D polyacrylamide gel electrophoresis proteomics approach, the differences in expressed brain proteome and phosphoproteome between 6-month-old PINK1-deficient mice and wild-type mice were identified. The observed changes in the brain proteome and phosphoproteome of mice lacking PINK1 suggest that defects in signaling networks, energy metabolism, cellular proteostasis, and neuronal structure and plasticity are involved in the pathogenesis of familial PD. Mutations in PINK1 are associated with an early-onset form of Parkinson's disease (PD). This study examines changes in the proteome and phosphoproteome of the PINK1 knockout mouse brain. Alterations were noted in several key proteins associated with: increased oxidative stress, aberrant cellular signaling, altered neuronal structure, decreased synaptic plasticity, reduced neurotransmission, diminished proteostasis networks, and altered metabolism. 14-3-3ε, 14-3-3 protein epsilon; 3-PGDH, phosphoglycerate dehydrogenase; ALDOA, aldolase A; APT1, acyl-protein thioesterase 1; CaM, calmodulin; CBR3, carbonyl reductase [NADPH] 3; ENO2, gamma-enolase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; HSP70

  8. Quantitative Phosphoproteomics of the Ataxia Telangiectasia-Mutated (ATM) and Ataxia Telangiectasia-Mutated and Rad3-related (ATR) Dependent DNA Damage Response in Arabidopsis thaliana*

    PubMed Central

    Roitinger, Elisabeth; Hofer, Manuel; Köcher, Thomas; Pichler, Peter; Novatchkova, Maria; Yang, Jianhua; Schlögelhofer, Peter; Mechtler, Karl

    2015-01-01

    The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions. They are conserved across eukaryotes, mediating the activation of cell-cycle checkpoints, chromatin modifications, and regulation of DNA repair proteins. We designed a novel mass spectrometry-based phosphoproteomics approach to study DNA damage repair in Arabidopsis thaliana. The protocol combines filter aided sample preparation, immobilized metal affinity chromatography, metal oxide affinity chromatography, and strong cation exchange chromatography for phosphopeptide generation, enrichment, and separation. Isobaric labeling employing iTRAQ (isobaric tags for relative and absolute quantitation) was used for profiling the phosphoproteome of atm atr double mutants and wild type plants under either regular growth conditions or challenged by irradiation. A total of 10,831 proteins were identified and 15,445 unique phosphopeptides were quantified, containing 134 up- and 38 down-regulated ATM/ATR dependent phosphopeptides. We identified known and novel ATM/ATR targets such as LIG4 and MRE11 (needed for resistance against ionizing radiation), PIE1 and SDG26 (implicated in chromatin remodeling), PCNA1, WAPL, and PDS5 (implicated in DNA replication), and ASK1 and HTA10 (involved in meiosis). PMID:25561503

  9. Quantitative Phosphoproteomics Reveals Pathways for Coordination of Cell Growth and Division by the Conserved Fission Yeast Kinase Pom1*

    PubMed Central

    Kettenbach, Arminja N.; Deng, Lin; Wu, Youjun; Baldissard, Suzanne; Adamo, Mark E.; Gerber, Scott A.; Moseley, James B.

    2015-01-01

    Complex phosphorylation-dependent signaling networks underlie the coordination of cellular growth and division. In the fission yeast Schizosaccharomyces pombe, the Dual specificity tyrosine-(Y)-phosphorylation regulated kinase (DYRK) family protein kinase Pom1 regulates cell cycle progression through the mitotic inducer Cdr2 and controls cell polarity through unknown targets. Here, we sought to determine the phosphorylation targets of Pom1 kinase activity by SILAC-based phosphoproteomics. We defined a set of high-confidence Pom1 targets that were enriched for cytoskeletal and cell growth functions. Cdr2 was the only cell cycle target of Pom1 kinase activity that we identified in cells. Mutation of Pom1-dependent phosphorylation sites in the C terminus of Cdr2 inhibited mitotic entry but did not impair Cdr2 localization. In addition, we found that Pom1 phosphorylated multiple substrates that function in polarized cell growth, including Tea4, Mod5, Pal1, the Rho GAP Rga7, and the Arf GEF Syt22. Purified Pom1 phosphorylated these cell polarity targets in vitro, confirming that they are direct substrates of Pom1 kinase activity and likely contribute to regulation of polarized growth by Pom1. Our study demonstrates that Pom1 acts in a linear pathway to control cell cycle progression while regulating a complex network of cell growth targets. PMID:25720772

  10. SILAC for global phosphoproteomic analysis.

    PubMed

    Pimienta, Genaro; Chaerkady, Raghothama; Pandey, Akhilesh

    2009-01-01

    Establishing the phosphorylation pattern of proteins in a comprehensive fashion is an important goal of a majority of cell signaling projects. Phosphoproteomic strategies should be designed in such a manner as to identify sites of phosphorylation as well as to provide quantitative information about the extent of phosphorylation at the sites. In this chapter, we describe an experimental strategy that outlines such an approach using stable isotope labeling with amino acids in cell culture (SILAC) coupled to LC-MS/MS. We highlight the importance of quantitative strategies in signal transduction as a platform for a systematic and global elucidation of biological processes.

  11. Quantitative phosphoproteomics reveals genistein as a modulator of cell cycle and DNA damage response pathways in triple-negative breast cancer cells

    PubMed Central

    FANG, YI; ZHANG, QIAN; WANG, XIN; YANG, XUE; WANG, XIANGYU; HUANG, ZHEN; JIAO, YUCHEN; WANG, JING

    2016-01-01

    Around one sixth of breast cancer cases are classified as triple-negative breast cancer (TNBC), named after the absence of the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2); however, patients with TNBC suffer from poor clinical outcome and shortage of targeted therapy. Genistein, an estrogenic soy isoflavone, shows anticancer effects in TNBC cells such as inducing G2/M cell cycle arrest and apoptosis. However, the underlying mechanism of its anticancer effects is poorly understood and its elucidation can help the development of novel therapeutic strategies for TNBC. In this study, by combining isobaric tag-based TMT labeling with titanium dioxide-based phosphopeptide enrichment, we quantitated 5,445 phosphorylation sites on 2,008 phosphoproteins in the TNBC cell line MDA-MB-231, upon genistein treatment. Our analysis revealed 332 genistein-regulated phosphorylation sites on 226 proteins. Our data show that genistein can regulate several biological processes during the cell cycle, including DNA replication, cohesin complex cleavage, and kinetochore formation. Furthermore, genistein can also activate DNA damage response, including activation of ATR and BRCA1 complex. Overall, our study presents evidence at a phosphoproteomic level that genistein is able to inhibit TNBC cell growth by regulating the cell cycle and DNA damage response in a more complex manner. Our findings help elucidate the mechanisms through which genistein exerts its anticancer effects in TNBC cells. PMID:26783066

  12. Quantitative Phosphoproteomics Analysis Reveals a Key Role of Insulin Growth Factor 1 Receptor (IGF1R) Tyrosine Kinase in Human Sperm Capacitation*

    PubMed Central

    Wang, Jing; Qi, Lin; Huang, Shaoping; Zhou, Tao; Guo, Yueshuai; Wang, Gaigai; Guo, Xuejiang; Zhou, Zuomin; Sha, Jiahao

    2015-01-01

    One of the most important changes during sperm capacitation is the enhancement of tyrosine phosphorylation. However, the mechanisms of protein tyrosine phosphorylation during sperm capacitation are not well studied. We used label-free quantitative phosphoproteomics to investigate the overall phosphorylation events during sperm capacitation in humans and identified 231 sites with increased phosphorylation levels. Motif analysis using the NetworKIN algorithm revealed that the activity of tyrosine phosphorylation kinases insulin growth factor 1 receptor (IGF1R)/insulin receptor is significantly enriched among the up-regulated phosphorylation substrates during capacitation. Western blotting further confirmed inhibition of IGF1R with inhibitors GSK1904529A and NVP-AEW541, which inhibited the increase in tyrosine phosphorylation levels during sperm capacitation. Additionally, sperm hyperactivated motility was also inhibited by GSK1904529A and NVP-AEW541 but could be up-regulated by insulin growth factor 1, the ligand of IGF1R. Thus, the IGF1R-mediated tyrosine phosphorylation pathway may play important roles in the regulation of sperm capacitation in humans and could be a target for improvement in sperm functions in infertile men. PMID:25693802

  13. Quantitative and Functional Phosphoproteomic Analysis Reveals that Ethylene Regulates Water Transport via the C-Terminal Phosphorylation of Aquaporin PIP2;1 in Arabidopsis.

    PubMed

    Qing, Dongjin; Yang, Zhu; Li, Mingzhe; Wong, Wai Shing; Guo, Guangyu; Liu, Shichang; Guo, Hongwei; Li, Ning

    2016-01-04

    Ethylene participates in the regulation of numerous cellular events and biological processes, including water loss, during leaf and flower petal wilting. The diverse ethylene responses may be regulated via dynamic interplays between protein phosphorylation/dephosphorylation and ubiquitin/26S proteasome-mediated protein degradation and protease cleavage. To address how ethylene alters protein phosphorylation through multi-furcated signaling pathways, we performed a (15)N stable isotope labelling-based, differential, and quantitative phosphoproteomics study on air- and ethylene-treated ethylene-insensitive Arabidopsis double loss-of-function mutant ein3-1/eil1-1. Among 535 non-redundant phosphopeptides identified, two and four phosphopeptides were up- and downregulated by ethylene, respectively. Ethylene-regulated phosphorylation of aquaporin PIP2;1 is positively correlated with the water flux rate and water loss in leaf. Genetic studies in combination with quantitative proteomics, immunoblot analysis, protoplast swelling/shrinking experiments, and leaf water loss assays on the transgenic plants expressing both the wild-type and S280A/S283A-mutated PIP2;1 in the both Col-0 and ein3eil1 genetic backgrounds suggest that ethylene increases water transport rate in Arabidopsis cells by enhancing S280/S283 phosphorylation at the C terminus of PIP2;1. Unknown kinase and/or phosphatase activities may participate in the initial up-regulation independent of the cellular functions of EIN3/EIL1. This finding contributes to our understanding of ethylene-regulated leaf wilting that is commonly observed during post-harvest storage of plant organs.

  14. Dissecting Dissection.

    ERIC Educational Resources Information Center

    AV Magazine, 1996

    1996-01-01

    This journal features articles covering various aspects of dissection. "Biology--The Study of Life" (George Russell) offers students experiments that do not require using invasive procedures. "Animal Cruelty--Behind the Scenes" (Zoe Weil) describes sources of laboratory animals. "Doing without Dissection" (Juliana…

  15. Integrated genomics and molecular breeding approaches for dissecting the complex quantitative traits in crop plants.

    PubMed

    Kujur, Alice; Saxena, Maneesha S; Bajaj, Deepak; Laxmi; Parida, Swarup K

    2013-12-01

    The enormous population growth, climate change and global warming are now considered major threats to agriculture and world's food security. To improve the productivity and sustainability of agriculture, the development of highyielding and durable abiotic and biotic stress-tolerant cultivars and/climate resilient crops is essential. Henceforth, understanding the molecular mechanism and dissection of complex quantitative yield and stress tolerance traits is the prime objective in current agricultural biotechnology research. In recent years, tremendous progress has been made in plant genomics and molecular breeding research pertaining to conventional and next-generation whole genome, transcriptome and epigenome sequencing efforts, generation of huge genomic, transcriptomic and epigenomic resources and development of modern genomics-assisted breeding approaches in diverse crop genotypes with contrasting yield and abiotic stress tolerance traits. Unfortunately, the detailed molecular mechanism and gene regulatory networks controlling such complex quantitative traits is not yet well understood in crop plants. Therefore, we propose an integrated strategies involving available enormous and diverse traditional and modern -omics (structural, functional, comparative and epigenomics) approaches/resources and genomics-assisted breeding methods which agricultural biotechnologist can adopt/utilize to dissect and decode the molecular and gene regulatory networks involved in the complex quantitative yield and stress tolerance traits in crop plants. This would provide clues and much needed inputs for rapid selection of novel functionally relevant molecular tags regulating such complex traits to expedite traditional and modern marker-assisted genetic enhancement studies in target crop species for developing high-yielding stress-tolerant varieties.

  16. Quantitative phosphoproteomics after auxin-stimulated lateral root induction identifies an SNX1 protein phosphorylation site required for growth.

    PubMed

    Zhang, Hongtao; Zhou, Houjiang; Berke, Lidija; Heck, Albert J R; Mohammed, Shabaz; Scheres, Ben; Menke, Frank L H

    2013-05-01

    Protein phosphorylation is instrumental to early signaling events. Studying system-wide phosphorylation in relation to processes under investigation requires a quantitative proteomics approach. In Arabidopsis, auxin application can induce pericycle cell divisions and lateral root formation. Initiation of lateral root formation requires transcriptional reprogramming following auxin-mediated degradation of transcriptional repressors. The immediate early signaling events prior to this derepression are virtually uncharacterized. To identify the signal molecules responding to auxin application, we used a lateral root-inducible system that was previously developed to trigger synchronous division of pericycle cells. To identify and quantify the early signaling events following this induction, we combined (15)N-based metabolic labeling and phosphopeptide enrichment and applied a mass spectrometry-based approach. In total, 3068 phosphopeptides were identified from auxin-treated root tissue. This root proteome dataset contains largely phosphopeptides not previously reported and represents one of the largest quantitative phosphoprotein datasets from Arabidopsis to date. Key proteins responding to auxin treatment included the multidrug resistance-like and PIN2 auxin carriers, auxin response factor2 (ARF2), suppressor of auxin resistance 3 (SAR3), and sorting nexin1 (SNX1). Mutational analysis of serine 16 of SNX1 showed that overexpression of the mutated forms of SNX1 led to retarded growth and reduction of lateral root formation due to the reduced outgrowth of the primordium, showing proof of principle for our approach.

  17. Quantitative Tools for Dissection of Hydrogen-Producing Metabolic Networks-Final Report

    SciTech Connect

    Rabinowitz, Joshua D.; Dismukes, G.Charles.; Rabitz, Herschel A.; Amador-Noguez, Daniel

    2012-10-19

    During this project we have pioneered the development of integrated experimental-computational technologies for the quantitative dissection of metabolism in hydrogen and biofuel producing microorganisms (i.e. C. acetobutylicum and various cyanobacteria species). The application of these new methodologies resulted in many significant advances in the understanding of the metabolic networks and metabolism of these organisms, and has provided new strategies to enhance their hydrogen or biofuel producing capabilities. As an example, using mass spectrometry, isotope tracers, and quantitative flux-modeling we mapped the metabolic network structure in C. acetobutylicum. This resulted in a comprehensive and quantitative understanding of central carbon metabolism that could not have been obtained using genomic data alone. We discovered that biofuel production in this bacterium, which only occurs during stationary phase, requires a global remodeling of central metabolism (involving large changes in metabolite concentrations and fluxes) that has the effect of redirecting resources (carbon and reducing power) from biomass production into solvent production. This new holistic, quantitative understanding of metabolism is now being used as the basis for metabolic engineering strategies to improve solvent production in this bacterium. In another example, making use of newly developed technologies for monitoring hydrogen and NAD(P)H levels in vivo, we dissected the metabolic pathways for photobiological hydrogen production by cyanobacteria Cyanothece sp. This investigation led to the identification of multiple targets for improving hydrogen production. Importantly, the quantitative tools and approaches that we have developed are broadly applicable and we are now using them to investigate other important biofuel producers, such as cellulolytic bacteria.

  18. Databases for plant phosphoproteomics.

    PubMed

    Schulze, Waltraud X; Yao, Qiuming; Xu, Dong

    2015-01-01

    Phosphorylation is the most studied posttranslational modification involved in signal transduction in stress responses, development, and growth. In the recent years large-scale phosphoproteomic studies were carried out using various model plants and several growth and stress conditions. Here we present an overview of online resources for plant phosphoproteomic databases: PhosPhAt as a resource for Arabidopsis phosphoproteins, P3DB as a resource expanding to crop plants, and Medicago PhosphoProtein Database as a resource for the model plant Medicago trunculata.

  19. Phosphoproteomics in cereals.

    PubMed

    Yang, Pingfang

    2015-01-01

    Cereals are the most important crop plant supplying staple food throughout the world. The economic importance and continued breeding of crop plants such as rice, maize, wheat, or barley require a detailed scientific understanding of adaptive and developmental processes. Protein phosphorylation is one of the most important regulatory posttranslational modifications and its analysis allows deriving functional and regulatory principles in plants. This minireview summarizes the current knowledge of phosphoproteomic studies in cereals.

  20. Acute Aortic Dissection Biomarkers Identified Using Isobaric Tags for Relative and Absolute Quantitation

    PubMed Central

    Xiao, Ziya; Xue, Yuan; Gu, Guorong; Zhang, Yaping; Zhang, Jin; Fan, Fan; Luan, Xiao; Deng, Zhi; Tao, Zhengang; Song, Zhen-ju; Tong, Chaoyang; Wang, Haojun

    2016-01-01

    The purpose of this study was to evaluate the utility of potential serum biomarkers for acute aortic dissection (AAD) that were identified by isobaric Tags for Relative and Absolute Quantitation (iTRAQ) approaches. Serum samples from 20 AAD patients and 20 healthy volunteers were analyzed using iTRAQ technology. Protein validation was performed using samples from 120 patients with chest pain. A total of 355 proteins were identified with the iTRAQ approach; 164 proteins reached the strict quantitative standard, and 125 proteins were increased or decreased more than 1.2-fold (64 and 61 proteins were up- and downregulated, resp.). Lumican, C-reactive protein (CRP), thrombospondin-1 (TSP-1), and D-dimer were selected as candidate biomarkers for the validation tests. Receiver operating characteristic (ROC) curves show that Lumican and D-dimer have diagnostic value (area under the curves [AUCs] 0.895 and 0.891, P < 0.05). For Lumican, the diagnostic sensitivity and specificity were 73.33% and 98.33%, while the corresponding values for D-dimer were 93.33% and 68.33%. For Lumican and D-dimer AAD combined diagnosis, the sensitivity and specificity were 88.33% and 95%, respectively. In conclusion, Lumican has good specificity and D-dimer has good sensitivity for the diagnosis of AAD, while the combined detection of D-dimer and Lumican has better diagnostic value. PMID:27403433

  1. Biphasic Affinity Chromatographic Approach for Deep Tyrosine Phosphoproteome Analysis.

    PubMed

    Deng, Zhenzhen; Dong, Mingming; Wang, Yan; Dong, Jing; Li, Shawn S-C; Zou, Hanfa; Ye, Mingliang

    2017-02-21

    Tyrosine phosphorylation (pTyr) is important for normal physiology and implicated in many human diseases, particularly cancer. Identification of pTyr sites is critical to dissecting signaling pathways and understanding disease pathologies. However, compared with serine/threonine phosphorylation (pSer/pThr), the analysis of pTyr at the proteome level is more challenging due to its low abundance. Here, we developed a biphasic affinity chromatographic approach where Src SH2 superbinder was coupled with NeutrAvidin affinity chromatography, for tyrosine phosphoproteome analysis. With the use of competitive elution agent biotin-pYEEI, this strategy can distinguish high-affinity phosphotyrosyl peptides from low-affinity ones, while the excess competitive agent is readily removed by using NeutrAvidin agarose resin in an integrated tip system. The excellent performance of this system was demonstrated by analyzing tyrosine phosphoproteome of Jurkat cells from which 3,480 unique pTyr sites were identified. The biphasic affinity chromatography method for deep Tyr phosphoproteome analysis is rapid, sensitive, robust, and cost-effective. It is widely applicable to the global analysis of the tyrosine phosphoproteome associated with tyrosine kinase signal transduction.

  2. Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation

    PubMed Central

    Nukarinen, Ella; Nägele, Thomas; Pedrotti, Lorenzo; Wurzinger, Bernhard; Mair, Andrea; Landgraf, Ramona; Börnke, Frederik; Hanson, Johannes; Teige, Markus; Baena-Gonzalez, Elena; Dröge-Laser, Wolfgang; Weckwerth, Wolfram

    2016-01-01

    Since years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a snrk1α1 knock out background (snrk1α1/α2) to abolish SnRK1 activity to understand major systemic functions of SnRK1 signalling under energy deprivation triggered by extended night treatment. We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants. PMID:27545962

  3. Phosphoproteomic profiling of the myocyte.

    PubMed

    Edwards, Alistair V G; Cordwell, Stuart J; White, Melanie Y

    2011-10-01

    Protein phosphorylation underpins major cellular processes including energy metabolism, signal transduction, excitation-contraction coupling, apoptosis, and cell survival mechanisms and is thus critical to the myocyte. Targeted approaches, whereby a handful of phosphoproteins are investigated, can suffer from a relatively narrow view of cellular phosphorylation. In contrast, recent technical advances have allowed for the comprehensive documentation of phosphorylation events in complex biological environments, providing a deeper view of the "phosphoproteome." A global, high-throughput characterization of the myocardial phosphoproteome, however, has not yet been achieved. Efficient analysis of phosphorylated proteins and their roles in a dynamic cellular environment requires high-resolution strategies that can identify, localize, and quantify many thousands of phosphorylation sites in a single experiment. Such an approach requires specific enrichment and purification techniques, developed to align with high-end instrumentation for analysis. Cutting-edge phosphoproteomics is no longer restricted to gel-based technology, instead focusing on affinity enrichment prior to liquid chromatography and mass spectrometry. We will describe the best current methods and how they can be applied, as well as the challenges associated with them. We also present current phosphoproteomic investigations in the myocyte and its subcompartments. Although the techniques and instrumentation required to achieve the goal of a myocardial phosphoprotein catalog in physiological and diseased states are highly specialized, the potential biological insight provided by such an approach makes phosphoproteomics an important new avenue of investigation for the cardiovascular researcher.

  4. Machine learning of global phosphoproteomic profiles enables discrimination of direct versus indirect kinase substrates.

    PubMed

    Kanshin, Evgeny; Giguere, Sebastien; Cheng, Jing; Tyers, Michael D; Thibault, Pierre

    2017-03-06

    Mass spectrometry allows quantification of tens of thousands of phosphorylation sites from minute amounts of cellular material. Despite this wealth of information, our understanding of phosphorylation-based signaling is limited, in part because it is not possible to deconvolute substrate phosphorylation that is directly mediated by a particular kinase versus phosphorylation that is mediated by downstream kinases. Here, we describe a framework for assignment of direct in-vivo kinase substrates using a combination of selective chemical inhibition, quantitative phosphoproteomics, and machine learning techniques. Our workflow allows classification of phosphorylation events following inhibition of an analog-sensitive kinase into kinase-independent effects of the inhibitor, direct effects on cognate substrates and indirect effects mediated by downstream kinases or phosphatases. We applied this method to identify many direct targets of Cdc28 and Snf1 kinases in the budding yeast S. cerevisiae. Global phosphoproteome analysis of acute time-series demonstrated that dephosphorylation of direct kinase substrates occurs more rapidly compared to indirect substrates, both after inhibitor treatment and under a physiological nutrient shift in wild-type cells. Mutagenesis experiments revealed a high proportion of functionally relevant phosphorylation sites on Snf1 targets. For example, Snf1 itself was inhibited through autophosphorylation on S391 and new phosphosites were discovered that modulate the activity of the Reg1 regulatory subunit of the Glc7 phosphatase and the Gal83 β-subunit of SNF1 complex. This methodology applies to any kinase for which a functional analog sensitive version can be constructed to facilitate the dissection of the global phosphorylation network.

  5. Insights Regarding Fungal Phosphoproteomic Analysis.

    PubMed

    Ribeiro, Liliane F C; Chelius, Cynthia L; Harris, Steven D; Marten, Mark R

    2017-03-10

    Protein phosphorylation is a major means of regulation for cellular processes, and is important in cell signaling, growth, and cell proliferation. To study phosphorylated proteins, high throughput phosphoproteomic technologies, such as reverse phase protein array, phospho-specific flow cytometry, and mass spectrometry (MS) based technologies, have been developed. Among them, mass spectrometry has become the primary tool employed for the identification of phosphoproteins and phosphosites in fungi, leading to an improved understanding of a number of signaling pathways. Using mass spectrometry techniques, researchers have discovered new kinase substrates, established connections between kinases and fungal pathogenicity, and studied the evolutionary lineage of kinases between different fungal species. Further, many specific phosphorylation sites recognized by individual kinases have been described. In this review, we will focus on recent discoveries made in yeast and filamentous fungi using phosphoproteomic analysis.

  6. Dissecting Analogical Leveling Quantitatively: The Case of the Innovative Potential Suffix in Tokyo Japanese.

    ERIC Educational Resources Information Center

    Matsuda, Kenjiro

    1993-01-01

    Analogical leveling in progress of a potential suffix in Tokyo Japanese is analyzed within a quantitative model. The phenomenon is shown to be controlled by five factors: sociological variable complex, verb stem length, verb conjugation pattern, the following inflectional form, and embeddedness of the clause containing the suffix. (Contains 70…

  7. Dissecting Quantitative Trait Loci for Boron Efficiency across Multiple Environments in Brassica napus

    PubMed Central

    Zhao, Zunkang; Wu, Likun; Nian, Fuzhao; Ding, Guangda; Shi, Taoxiong; Zhang, Didi; Shi, Lei; Xu, Fangsen; Meng, Jinling

    2012-01-01

    High yield is the most important goal in crop breeding, and boron (B) is an essential micronutrient for plants. However, B deficiency, leading to yield decreases, is an agricultural problem worldwide. Brassica napus is one of the most sensitive crops to B deficiency, and considerable genotypic variation exists among different cultivars in response to B deficiency. To dissect the genetic basis of tolerance to B deficiency in B. napus, we carried out QTL analysis for seed yield and yield-related traits under low and normal B conditions using the double haploid population (TNDH) by two-year and the BQDH population by three-year field trials. In total, 80 putative QTLs and 42 epistatic interactions for seed yield, plant height, branch number, pod number, seed number, seed weight and B efficiency coefficient (BEC) were identified under low and normal B conditions, singly explaining 4.15–23.16% and 0.53–14.38% of the phenotypic variation. An additive effect of putative QTLs was a more important controlling factor than the additive-additive effect of epistatic interactions. Four QTL-by-environment interactions and 7 interactions between epistatic interactions and the environment contributed to 1.27–4.95% and 1.17–3.68% of the phenotypic variation, respectively. The chromosome region on A2 of SYLB-A2 for seed yield under low B condition and BEC-A2 for BEC in the two populations was equivalent to the region of a reported major QTL, BE1. The B. napus homologous genes of Bra020592 and Bra020595 mapped to the A2 region and were speculated to be candidate genes for B efficiency. These findings reveal the complex genetic basis of B efficiency in B. napus. They provide a basis for the fine mapping and cloning of the B efficiency genes and for breeding B-efficient cultivars by marker-assisted selection (MAS). PMID:23028855

  8. Functional phosphoproteomic mass spectrometry-based approaches

    PubMed Central

    2012-01-01

    Mass Spectrometry (MS)-based phosphoproteomics tools are crucial for understanding the structure and dynamics of signaling networks. Approaches such as affinity purification followed by MS have also been used to elucidate relevant biological questions in health and disease. The study of proteomes and phosphoproteomes as linked systems, rather than research studies of individual proteins, are necessary to understand the functions of phosphorylated and un-phosphorylated proteins under spatial and temporal conditions. Phosphoproteome studies also facilitate drug target protein identification which may be clinically useful in the near future. Here, we provide an overview of general principles of signaling pathways versus phosphorylation. Likewise, we detail chemical phosphoproteomic tools, including pros and cons with examples where these methods have been applied. In addition, basic clues of electrospray ionization and collision induced dissociation fragmentation are detailed in a simple manner for successful phosphoproteomic clinical studies. PMID:23369623

  9. Dissecting eukaryotic cells by coherent phase microscopy: quantitative analysis of quiescent and activated T lymphocytes

    NASA Astrophysics Data System (ADS)

    Tychinsky, Vladimir P.; Kretushev, Alexander V.; Vyshenskaya, Tatiana V.; Shtil, Alexander A.

    2012-07-01

    We present a concept for quantitative characterization of a functional state of an individual eukaryotic cell based on interference imaging. The informative parameters of the phase images of quiescent and mitogen-activated T lymphocytes included the phase thickness, phase volume, the area, and the size of organelles. These parameters were obtained without a special hypothesis about cell structure. Combinations of these parameters generated a ``phase portrait'' of the cell. A simplified spherical multilayer optic model of a T lymphocyte was used to calculate the refractivity profile, to identify structural elements of the image with the organelles, and to interpret the parameters of the phase portrait. The values of phase image parameters underwent characteristic changes in the course of mitogenic stimulation of T cells; thereby, the functional state of individual cells can be described using these parameters. Because the values of the components of the phase portrait are measured in absolute units, it is possible to compare the parameters of images obtained with different interference microscopes. Thus, the analysis of phase portraits provides a new and perspective approach for quantitative, real-time analysis of subcellular structure and physiologic state of an individual cell.

  10. Quantitative proteomic dissection of a native 14-3-3ε interacting protein complex associated with hepatocellular carcinoma.

    PubMed

    Bai, Chen; Tang, Siwei; Bai, Chen; Chen, Xian

    2014-04-01

    The 14-3-3 proteins regulate diverse biological processes that are implicated in cancer development, and seven 14-3-3 isoforms were identified with isoform-specific roles in different human tumors. In our previous work, we dissected the interactome of 14-3-3ε formed during the DNA damage response in a hepatocellular carcinoma (HCC) cell using an AACT/SILAC-based quantitative proteomic approach. In this study, we used a similar proteomic approach to profile/identify the 14-3-3ε interactome formed in native HCC cells. Functional categorization and data-dependent network analysis of the native HCC-specific 14-3-3ε interactome revealed that 14-3-3ε is involved in the regulation of multiple biological processes (BPs)/pathways, including cell cycle control, apoptosis, signal transduction, transport, cell adhesion, carbohydrate metabolism, and nucleic acid metabolism. Biological validation further supports that 14-3-3ε, via association with multiple BP/pathway-specific proteins, coordinates the regulation of proliferation, survival, and metastasis of HCC. The findings in this study, together with those of our previous study, provide an extensive profile of the 14-3-3ε interaction network in HCC cells, which should be valuable for understanding the pathology of HCC and HCC therapy.

  11. Glucose-regulated and drug-perturbed phosphoproteome reveals molecular mechanisms controlling insulin secretion

    PubMed Central

    Sacco, Francesca; Humphrey, Sean J.; Cox, Jürgen; Mischnik, Marcel; Schulte, Anke; Klabunde, Thomas; Schäfer, Matthias; Mann, Matthias

    2016-01-01

    Insulin-secreting beta cells play an essential role in maintaining physiological blood glucose levels, and their dysfunction leads to the development of diabetes. To elucidate the signalling events regulating insulin secretion, we applied a recently developed phosphoproteomics workflow. We quantified the time-resolved phosphoproteome of murine pancreatic cells following their exposure to glucose and in combination with small molecule compounds that promote insulin secretion. The quantitative phosphoproteome of 30,000 sites clustered into three main groups in concordance with the modulation of the three key kinases: PKA, PKC and CK2A. A high-resolution time course revealed key novel regulatory sites, revealing the importance of methyltransferase DNMT3A phosphorylation in the glucose response. Remarkably a significant proportion of these novel regulatory sites is significantly downregulated in diabetic islets. Control of insulin secretion is embedded in an unexpectedly broad and complex range of cellular functions, which are perturbed by drugs in multiple ways. PMID:27841257

  12. Phosphoproteomics for the masses

    PubMed Central

    Grimsrud, Paul A.; Swaney, Danielle L.; Wenger, Craig D.; Beauchene, Nicole A.; Coon, Joshua J.

    2010-01-01

    Protein phosphorylation serves as a primary mechanism of signal transduction in the cells of biological organisms. Technical advancements over the last several years in mass spectrometry now allow for the large-scale identification and quantitation of in vivo phosphorylation at unprecedented levels. These developments have occurred in the areas of sample preparation, instrumentation, quantitative methodology, and informatics so that today, ten to twenty thousand phosphorylation sites can be identified and quantified within a few weeks. With the rapid development and widespread availability of such data, its translation into biological insight and knowledge is a current obstacle. Here we present an overview of how this technology came to be and is currently applied, as well as future challenges for the field. PMID:20047291

  13. Quantitative trait loci mapping and genetic dissection for lint percentage in upland cotton (Gossypium hirsutum).

    PubMed

    Wang, Min; Li, Chengqi; Wang, Qinglian

    2014-08-01

    Lint percentage is an important character of cotton yield components and it is also correlated with cotton fibre development. In this study, we used a high lint percentage variety, Baimian1, and a low lint percentage, TM-1 genetic standard for Gossypium hirsutum, as parents to construct a mapping populations in upland cotton (G. hirsutum). A quantitative trait locus/loci (QTL) analysis of lint percentage was performed by using two mapping procedures; composite interval mapping (CIM), inclusive composite interval mapping (ICIM) and the F2:3 populations in 2 years. Six main-effect QTL (M-QTL) for lint percentage (four significant and two suggestive) were detected in both years by CIM, and were located on chr. 3, chr. 19, chr. 26 and chr. 5/chr. 19. Of the six QTL, marker intervals and favourable gene sources of the significant M-QTL, qLP-3(2010) and qLP-3(2011) were consistent. These QTL were also detected by ICIM, and therefore, should preferentially be used for markerassisted selection (MAS) of lint percentage. Another M-QTL, qLP-19(2010), was detected by two mapping procedures, and it could also be a candidate for MAS. We detected the interaction between two M-QTL and environment, and 11 epistatic QTL (E-QTL) and their interaction with environment by using ICIM. The study also found two EST-SSRs, NAU1187 and NAU1255, linked to M-QTL for lint percentage that could be candidate markers affecting cotton fibre development.

  14. Genetic dissection of fruiting body-related traits using quantitative trait loci mapping in Lentinula edodes.

    PubMed

    Gong, Wen-Bing; Li, Lei; Zhou, Yan; Bian, Yin-Bing; Kwan, Hoi-Shan; Cheung, Man-Kit; Xiao, Yang

    2016-06-01

    To provide a better understanding of the genetic architecture of fruiting body formation of Lentinula edodes, quantitative trait loci (QTLs) mapping was employed to uncover the loci underlying seven fruiting body-related traits (FBRTs). An improved L. edodes genetic linkage map, comprising 572 markers on 12 linkage groups with a total map length of 983.7 cM, was constructed by integrating 82 genomic sequence-based insertion-deletion (InDel) markers into a previously published map. We then detected a total of 62 QTLs for seven target traits across two segregating testcross populations, with individual QTLs contributing 5.5 %-30.2 % of the phenotypic variation. Fifty-three out of the 62 QTLs were clustered in six QTL hotspots, suggesting the existence of main genomic regions regulating the morphological characteristics of fruiting bodies in L. edodes. A stable QTL hotspot on MLG2, containing QTLs for all investigated traits, was identified in both testcross populations. QTLs for related traits were frequently co-located on the linkage groups, demonstrating the genetic basis for phenotypic correlation of traits. Meta-QTL (mQTL) analysis was performed and identified 16 mQTLs with refined positions and narrow confidence intervals (CIs). Nine genes, including those encoding MAP kinase, blue-light photoreceptor, riboflavin-aldehyde-forming enzyme and cyclopropane-fatty-acyl-phospholipid synthase, and cytochrome P450s, were likely to be candidate genes controlling the shape of fruiting bodies. The study has improved our understanding of the genetic architecture of fruiting body formation in L. edodes. To our knowledge, this is the first genome-wide QTL detection of FBRTs in L. edodes. The improved genetic map, InDel markers and QTL hotspot regions revealed here will assist considerably in the conduct of future genetic and breeding studies of L. edodes.

  15. Phosphoproteome Discovery in Human Biological Fluids

    PubMed Central

    Giorgianni, Francesco; Beranova-Giorgianni, Sarka

    2016-01-01

    Phosphorylation plays a critical role in regulating protein function and thus influences a vast spectrum of cellular processes. With the advent of modern bioanalytical technologies, examination of protein phosphorylation on a global scale has become one of the major research areas. Phosphoproteins are found in biological fluids and interrogation of the phosphoproteome in biological fluids presents an exciting opportunity for discoveries that hold great potential for novel mechanistic insights into protein function in health and disease, and for translation to improved diagnostic and therapeutic approaches for the clinical setting. This review focuses on phosphoproteome discovery in selected human biological fluids: serum/plasma, urine, cerebrospinal fluid, saliva, and bronchoalveolar lavage fluid. Bioanalytical workflows pertinent to phosphoproteomics of biological fluids are discussed with emphasis on mass spectrometry-based approaches, and summaries of studies on phosphoproteome discovery in major fluids are presented. PMID:28248247

  16. Influence of sample preparation and reliability of automated numerical refocusing in stain-free analysis of dissected tissues with quantitative phase digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Lenz, Philipp; Bettenworth, Dominik; Krausewitz, Philipp; Domagk, Dirk; Ketelhut, Steffi

    2015-05-01

    Digital holographic microscopy (DHM) has been demonstrated to be a versatile tool for high resolution non-destructive quantitative phase imaging of surfaces and multi-modal minimally-invasive monitoring of living cell cultures in-vitro. DHM provides quantitative monitoring of physiological processes through functional imaging and structural analysis which, for example, gives new insight into signalling of cellular water permeability and cell morphology changes due to toxins and infections. Also the analysis of dissected tissues quantitative DHM phase contrast prospects application fields by stain-free imaging and the quantification of tissue density changes. We show that DHM allows imaging of different tissue layers with high contrast in unstained tissue sections. As the investigation of fixed samples represents a very important application field in pathology, we also analyzed the influence of the sample preparation. The retrieved data demonstrate that the quality of quantitative DHM phase images of dissected tissues depends strongly on the fixing method and common staining agents. As in DHM the reconstruction is performed numerically, multi-focus imaging is achieved from a single digital hologram. Thus, we evaluated the automated refocussing feature of DHM for application on different types of dissected tissues and revealed that on moderately stained samples highly reproducible holographic autofocussing can be achieved. Finally, it is demonstrated that alterations of the spatial refractive index distribution in murine and human tissue samples represent a reliable absolute parameter that is related of different degrees of inflammation in experimental colitis and Crohn's disease. This paves the way towards the usage of DHM in digital pathology for automated histological examinations and further studies to elucidate the translational potential of quantitative phase microscopy for the clinical management of patients, e.g., with inflammatory bowel disease.

  17. Comparative phosphoproteomics of zebrafish Fyn/Yes morpholino knockdown embryos.

    PubMed

    Lemeer, Simone; Jopling, Chris; Gouw, Joost; Mohammed, Shabaz; Heck, Albert J R; Slijper, Monique; den Hertog, Jeroen

    2008-11-01

    The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO(2)-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.

  18. Refined phosphopeptide enrichment by phosphate additive and the analysis of human brain phosphoproteome.

    PubMed

    Tan, Haiyan; Wu, Zhiping; Wang, Hong; Bai, Bing; Li, Yuxin; Wang, Xusheng; Zhai, Bo; Beach, Thomas G; Peng, Junmin

    2015-01-01

    Alzheimer's disease (AD) is the most common form of dementia, characterized by progressive loss of cognitive function. One of the pathological hallmarks of AD is the formation of neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein, but global deregulation of protein phosphorylation in AD is not well analyzed. Here, we report a pilot investigation of AD phosphoproteome by titanium dioxide enrichment coupled with high resolution LC-MS/MS. During the optimization of the enrichment method, we found that phosphate ion at a low concentration (e.g. 1 mM) worked efficiently as a nonphosphopeptide competitor to reduce background. The procedure was further tuned with respect to peptide-to-bead ratio, phosphopeptide recovery, and purity. Using this refined method and 9 h LC-MS/MS, we analyzed phosphoproteome in one milligram of digested AD brain lysate, identifying 5243 phosphopeptides containing 3715 nonredundant phosphosites on 1455 proteins, including 31 phosphosites on the tau protein. This modified enrichment method is simple and highly efficient. The AD case study demonstrates its feasibility of dissecting phosphoproteome in a limited amount of postmortem human brain. All MS data have been deposited in the ProteomeXchange with identifier PXD001180 (http://proteomecentral.proteomexchange.org/dataset/PXD001180).

  19. Technical phosphoproteomic and bioinformatic tools useful in cancer research

    PubMed Central

    2011-01-01

    Reversible protein phosphorylation is one of the most important forms of cellular regulation. Thus, phosphoproteomic analysis of protein phosphorylation in cells is a powerful tool to evaluate cell functional status. The importance of protein kinase-regulated signal transduction pathways in human cancer has led to the development of drugs that inhibit protein kinases at the apex or intermediary levels of these pathways. Phosphoproteomic analysis of these signalling pathways will provide important insights for operation and connectivity of these pathways to facilitate identification of the best targets for cancer therapies. Enrichment of phosphorylated proteins or peptides from tissue or bodily fluid samples is required. The application of technologies such as phosphoenrichments, mass spectrometry (MS) coupled to bioinformatics tools is crucial for the identification and quantification of protein phosphorylation sites for advancing in such relevant clinical research. A combination of different phosphopeptide enrichments, quantitative techniques and bioinformatic tools is necessary to achieve good phospho-regulation data and good structural analysis of protein studies. The current and most useful proteomics and bioinformatics techniques will be explained with research examples. Our aim in this article is to be helpful for cancer research via detailing proteomics and bioinformatic tools. PMID:21967744

  20. Phosphoproteomic Analysis of Aurora Kinase Inhibition in Monopolar Cytokinesis.

    PubMed

    Polat, Ayse Nur; Karayel, Özge; Giese, Sven H; Harmanda, Büşra; Sanal, Erdem; Hu, Chi-Kuo; Renard, Bernhard Y; Özlü, Nurhan

    2015-09-04

    Cytokinesis is the last step of the cell cycle that requires coordinated activities of the microtubule cytoskeleton, actin cytoskeleton, and membrane compartments. Aurora B kinase is one of the master regulatory kinases that orchestrate multiple events during cytokinesis. To reveal targets of the Aurora B kinase, we combined quantitative mass spectrometry with chemical genetics. Using the quantitative proteomic approach, SILAC (stable isotope labeling with amino acids in cell culture), we analyzed the phosphoproteome of monopolar cytokinesis upon VX680- or AZD1152-mediated aurora kinase inhibition. In total, our analysis quantified over 20 000 phosphopeptides in response to the Aurora-B kinase inhibition; 246 unique phosphopeptides were significantly down-regulated and 74 were up-regulated. Our data provide a broad analysis of downstream effectors of Aurora kinase and offer insights into how Aurora kinase regulates cytokinesis.

  1. Characterization of the Phosphoproteome in SLE Patients

    PubMed Central

    Huang, Jianrong; Dai, Yong

    2012-01-01

    Protein phosphorylation is a complex regulatory event that is involved in the signaling networks that affect virtually every cellular process. The protein phosphorylation may be a novel source for discovering biomarkers and drug targets. However, a systematic analysis of the phosphoproteome in patients with SLE has not been performed. To clarify the pathogenesis of systemic lupus erythematosus (SLE), we compared phosphoprotein expression in PBMCs from SLE patients and normal subjects using proteomics analyses. Phosphopeptides were enriched using TiO2 from PBMCs isolated from 15 SLE patients and 15 healthy subjects and then analyzed by automated LC-MS/MS analysis. Phosphorylation sites were identified and quantitated by MASCOT and MaxQuant. A total of 1035 phosphorylation sites corresponding to 618 NCBI-annotated genes were identified in SLE patients compared with normal subjects. Differentially expressed proteins, peptides and phosphorylation sites were then subjected to bioinformatics analyses. Gene ontology(GO) and pathway analyses showed that nucleic acid metabolism, cellular component organization, transport and multicellular organismal development pathways made up the largest proportions of the differentially expressed genes. Pathway analyses showed that the mitogen-activated protein kinase (MAPK) signaling pathway and actin cytoskeleton regulators made up the largest proportions of the metabolic pathways. Network analysis showed that rous sarcoma oncogene (SRC), v-rel reticuloendotheliosis viral oncogene homolog A (RELA), histone deacetylase (HDA1C) and protein kinase C, delta (PRKCD) play important roles in the stability of the network. These data suggest that aberrant protein phosphorylation may contribute to SLE pathogenesis. PMID:23285258

  2. Neck dissection

    MedlinePlus

    ... There are three main types of neck dissection surgery: Radical neck dissection: All the tissue on the side of ... Lund LJ, et al, eds. Cummings Otolaryngology: Head & Neck Surgery . 5th ed. Philadelphia, PA: Elsevier Mosby; 2010:chap ...

  3. Phosphoproteomic Analysis of Cell-Based Resistance to BRAF Inhibitor Therapy in Melanoma

    PubMed Central

    Parker, Robert; Vella, Laura J.; Xavier, Dylan; Amirkhani, Ardeshir; Parker, Jimmy; Cebon, Jonathan; Molloy, Mark P.

    2015-01-01

    The treatment of melanoma by targeted inhibition of the mutated kinase BRAF with small molecules only temporarily suppresses metastatic disease. In the face of chemical inhibition tumor plasticity, both innate and adaptive, promotes survival through the biochemical and genetic reconfiguration of cellular pathways that can engage proliferative and migratory systems. To investigate this process, high-resolution mass spectrometry was used to characterize the phosphoproteome of this transition in vitro. A simple and accurate, label-free quantitative method was used to localize and quantitate thousands of phosphorylation events. We also correlated changes in the phosphoproteome with the proteome to more accurately determine changes in the activity of regulatory kinases determined by kinase landscape profiling. The abundance of phosphopeptides with sites that function in cytoskeletal regulation, GTP/GDP exchange, protein kinase C, IGF signaling, and melanosome maturation were highly divergent after transition to a drug resistant phenotype. PMID:26029660

  4. Identifying differentially regulated subnetworks from phosphoproteomic data

    PubMed Central

    2010-01-01

    Background Various high throughput methods are available for detecting regulations at the level of transcription, translation or posttranslation (e.g. phosphorylation). Integrating these data with protein networks should make it possible to identify subnetworks that are significantly regulated. Furthermore, such integration can support identification of regulated entities from often noisy high throughput data. In particular, processing mass spectrometry-based phosphoproteomic data in this manner may expose signal transduction pathways and, in the case of experiments with drug-treated cells, reveal the drug's mode of action. Results Here, we introduce SubExtractor, an algorithm that combines phosphoproteomic data with protein network information from STRING to identify differentially regulated subnetworks and individual proteins. The method is based on a Bayesian probabilistic model combined with a genetic algorithm and rigorous significance testing. The Bayesian model accounts for information about both differential regulation and network topology. The method was tested with artificial data and subsequently applied to a comprehensive phosphoproteomics study investigating the mode of action of sorafenib, a small molecule kinase inhibitor. Conclusions SubExtractor reliably identifies differentially regulated subnetworks from phosphoproteomic data by integrating protein networks. The method can also be applied to gene or protein expression data. PMID:20584295

  5. TSLP Signaling Network Revealed by SILAC-Based Phosphoproteomics*

    PubMed Central

    Zhong, Jun; Kim, Min-Sik; Chaerkady, Raghothama; Wu, Xinyan; Huang, Tai-Chung; Getnet, Derese; Mitchell, Christopher J.; Palapetta, Shyam M.; Sharma, Jyoti; O'Meally, Robert N.; Cole, Robert N.; Yoda, Akinori; Moritz, Albrecht; Loriaux, Marc M.; Rush, John; Weinstock, David M.; Tyner, Jeffrey W.; Pandey, Akhilesh

    2012-01-01

    Thymic stromal lymphopoietin (TSLP) is a cytokine that plays diverse roles in the regulation of immune responses. TSLP requires a heterodimeric receptor complex consisting of IL-7 receptor α subunit and its unique TSLP receptor (gene symbol CRLF2) to transmit signals in cells. Abnormal TSLP signaling (e.g. overexpression of TSLP or its unique receptor TSLPR) contributes to the development of a number of diseases including asthma and leukemia. However, a detailed understanding of the signaling pathways activated by TSLP remains elusive. In this study, we performed a global quantitative phosphoproteomic analysis of the TSLP signaling network using stable isotope labeling by amino acids in cell culture. By employing titanium dioxide in addition to antiphosphotyrosine antibodies as enrichment methods, we identified 4164 phosphopeptides on 1670 phosphoproteins. Using stable isotope labeling by amino acids in cell culture-based quantitation, we determined that the phosphorylation status of 226 proteins was modulated by TSLP stimulation. Our analysis identified activation of several members of the Src and Tec families of kinases including Btk, Lyn, and Tec by TSLP for the first time. In addition, we report TSLP-induced phosphorylation of protein phosphatases such as Ptpn6 (SHP-1) and Ptpn11 (Shp2), which has also not been reported previously. Co-immunoprecipitation assays showed that Shp2 binds to the adapter protein Gab2 in a TSLP-dependent manner. This is the first demonstration of an inducible protein complex in TSLP signaling. A kinase inhibitor screen revealed that pharmacological inhibition of PI-3 kinase, Jak family kinases, Src family kinases or Btk suppressed TSLP-dependent cellular proliferation making them candidate therapeutic targets in diseases resulting from aberrant TSLP signaling. Our study is the first phosphoproteomic analysis of the TSLP signaling pathway that greatly expands our understanding of TSLP signaling and provides novel therapeutic targets

  6. [Aortic dissection].

    PubMed

    Ogino, Hitoshi

    2011-07-01

    Acute aortic dissection suddenly occurrs and results in a variety of catastrophic sequelae including cardiac tamponade, rupture, and organ malperfusion. In acute stage (< 2 weeks), according to the classifications on the region of aortic dissection, the condition of the false channel and the onset, appropriate medical, surgical, or endovascular treatments including endovascular aneurysm repair followed by the rapid and accurate diagnosis of aortic dissection using computed tomography and ultrasound should be performed without delay. In the chronic stage (> 2 weeks), the behavior of the chronic dissection or residual distal dissection after the initial treatment should be followed-up carefully with best medical treatment at the regular intervals. If necessary, appropriate surgical and endovascular treatment should be carried out in the proper timing before rupture.

  7. Dissecting the genetic components of a quantitative trait locus for blood pressure and renal pathology on rat chromosome 3

    PubMed Central

    Koh-Tan, H.H. Caline; Dashti, Mohammed; Wang, Ting; Beattie, Wendy; Mcclure, John; Young, Barbara; Dominiczak, Anna F.; McBride, Martin W.; Graham, Delyth

    2017-01-01

    Background: We have previously confirmed the importance of rat chromosome 3 (RNO3) genetic loci on blood pressure elevation, pulse pressure (PP) variability and renal pathology during salt challenge in the stroke-prone spontaneously hypertensive (SHRSP) rat. The aims of this study were to generate a panel of RNO3 congenic sub-strains to genetically dissect the implicated loci and identify positional candidate genes by microarray expression profiling and analysis of next-generation sequencing data. Method and results: A panel of congenic sub-strains were generated containing Wistar–Kyoto (WKY)-introgressed segments of varying size on the SHRSP genetic background, focused within the first 50 Mbp of RNO3. Haemodynamic profiling during salt challenge demonstrated significantly reduced systolic blood pressure, diastolic blood pressure and PP variability in SP.WKYGla3a, SP.WKYGla3c, SP.WKYGla3d and SP.WKYGla3e sub-strains. Only SBP and DBP were significantly reduced during salt challenge in SP.WKYGla3b and SP.WKYGla3f sub-strains, whereas SP.WKYGla3g rats did not differ in haemodynamic response to SHRSP. Those sub-strains demonstrating significantly reduced PP variability during salt challenge also demonstrated significantly reduced renal pathology and proteinuria. Microarray expression profiling prioritized two candidate genes for blood pressure regulation (Dnm1, Tor1b), localized within the common congenic interval shared by SP.WKYGla3d and SP.WKYGla3f strains, and one candidate gene for salt-induced PP variability and renal pathology (Rabgap1), located within the region unique to the SP.WKYGla3d strain. Comparison of next-generation sequencing data identified variants within additional positional genes that are likely to affect protein function. Conclusion: This study has identified distinct intervals on RNO3-containing genes that may be important for blood pressure regulation and renal pathology during salt challenge. PMID:27755386

  8. Genome-wide conserved non-coding microsatellite (CNMS) marker-based integrative genetical genomics for quantitative dissection of seed weight in chickpea.

    PubMed

    Bajaj, Deepak; Saxena, Maneesha S; Kujur, Alice; Das, Shouvik; Badoni, Saurabh; Tripathi, Shailesh; Upadhyaya, Hari D; Gowda, C L L; Sharma, Shivali; Singh, Sube; Tyagi, Akhilesh K; Parida, Swarup K

    2015-03-01

    Phylogenetic footprinting identified 666 genome-wide paralogous and orthologous CNMS (conserved non-coding microsatellite) markers from 5'-untranslated and regulatory regions (URRs) of 603 protein-coding chickpea genes. The (CT)n and (GA)n CNMS carrying CTRMCAMV35S and GAGA8BKN3 regulatory elements, respectively, are abundant in the chickpea genome. The mapped genic CNMS markers with robust amplification efficiencies (94.7%) detected higher intraspecific polymorphic potential (37.6%) among genotypes, implying their immense utility in chickpea breeding and genetic analyses. Seventeen differentially expressed CNMS marker-associated genes showing strong preferential and seed tissue/developmental stage-specific expression in contrasting genotypes were selected to narrow down the gene targets underlying seed weight quantitative trait loci (QTLs)/eQTLs (expression QTLs) through integrative genetical genomics. The integration of transcript profiling with seed weight QTL/eQTL mapping, molecular haplotyping, and association analyses identified potential molecular tags (GAGA8BKN3 and RAV1AAT regulatory elements and alleles/haplotypes) in the LOB-domain-containing protein- and KANADI protein-encoding transcription factor genes controlling the cis-regulated expression for seed weight in the chickpea. This emphasizes the potential of CNMS marker-based integrative genetical genomics for the quantitative genetic dissection of complex seed weight in chickpea.

  9. Phosphoproteomics technologies and applications in plant biology research

    PubMed Central

    Li, Jinna; Silva-Sanchez, Cecilia; Zhang, Tong; Chen, Sixue; Li, Haiying

    2015-01-01

    Protein phosphorylation has long been recognized as an essential mechanism to regulate many important processes of plant life. However, studies on phosphorylation mediated signaling events in plants are challenged with low stoichiometry and dynamic nature of phosphorylated proteins. Significant advances in mass spectrometry based phosphoproteomics have taken place in recent decade, including phosphoprotein/phosphopeptide enrichment, detection and quantification, and phosphorylation site localization. This review describes a variety of separation and enrichment methods for phosphoproteins and phosphopeptides, the applications of technological innovations in plant phosphoproteomics, and highlights significant achievement of phosphoproteomics in the areas of plant signal transduction, growth and development. PMID:26136758

  10. Genetic Dissection of a Blood Pressure Quantitative Trait Locus on Rat Chromosome 1 and Gene Expression Analysis Identifies SPON1 as a Novel Candidate Hypertension Gene

    PubMed Central

    Clemitson, Jenny-Rebecca; Dixon, Richard J.; Haines, Steve; Bingham, Andrew J.; Patel, Bhakti R.; Hall, Laurence; Lo, Ming; Sassard, Jean; Charchar, Fadi J.; Samani, Nilesh J.

    2007-01-01

    A region with a major effect on blood pressure is located on rat chromosome 1. We have previously isolated this region in reciprocal congenic strains (WKY.SHR-Sa and SHR.WKY-Sa) derived from a cross of the spontaneously hypertensive rat (SHR) with the Wistar-Kyoto rat (WKY) and shown that there are two distinct BP quantitative trait loci (QTLs), BP1 and BP2, in this region. Sisa1, a congenic sub-strain from the SHR.WKY-Sa animals carrying an introgressed segment of 4.3Mb, contains BP1. Here, we report further dissection of BP1 by the creation of two new mutually exclusive congenic sub-strains (Sisa1a and Sisa1b) and interrogation of candidate genes by expression profiling and targeted transcript sequencing. Only one of the sub-strains (Sisa1a) continued to demonstrate a BP difference but with a reduced introgressed segment of 3Mb. Exonic sequencing of the twenty genes located in the Sisa1a region did not identify any major differences between SHR and WKY. However, microarray expression profiling of whole kidney samples and subsequent quantitative RT-PCR identified a single gene, Spon1 that exhibited significant differential expression between the WKY and SHR genotypes at both 6 and 24 weeks of age. Western blot analysis confirmed an increased level of the Spon1 gene product in SHR kidneys. Spon1 belongs to a family of genes with anti-angiogenic properties. These findings justify further investigation of this novel positional candidate gene in BP control in hypertensive rat models and humans. PMID:17332427

  11. Applicability of a "speed" congenic strategy to dissect blood pressure quantitative trait loci on rat chromosome 2.

    PubMed

    Jeffs, B; Negrin, C D; Graham, D; Clark, J S; Anderson, N H; Gauguier, D; Dominiczak, A F

    2000-01-01

    The identification of any quantitative trait locus (QTL) via a genome scan is only the first step toward the ultimate goal of gene identification. The next step is the production of congenic strains by which the existence of a QTL may be verified and the implicated chromosomal region be reduced to a size applicable to positional cloning of the causal gene. We used a speed congenic breeding protocol previously verified in mice for 2 blood pressure QTLs on rat chromosome 2. Four congenic strains were produced through introgression of various segments of chromosome 2 from Wistar-Kyoto rats from Glasgow colonies [WKY((Gla)) rats] into the recipient stroke-prone spontaneously hypertensive rats from Glasgow colonies [SHRSP((Gla))], and vice versa. The number of backcross generations required for each strain to achieve complete homozygosity at 83 background genetic markers in a "best" male varied between 3 and 4. Transfer of the region of rat chromosome 2 containing both QTLs from WKY((Gla)) into an SHRSP((Gla)) genetic background lowered both baseline and salt-loaded systolic blood pressure by approximately 20 and approximately 40 mm Hg in male congenic rats compared with the SHRSP parental strain (F=53.4, P<0.005; F=28.0, P< 0.0005, respectively). In contrast, control animals for stowaway heterozygosity presented no deviation from the blood pressure values recorded for the SHRSP((Gla)), indicating that if such heterozygosity exists, its effect on blood pressure is negligible. A reciprocal strategy in which 1 or both QTLs on rat chromosome 2 were transferred from SHRSP((Gla)) into a WKY((Gla)) genetic background resulted in statistically significant but smaller blood pressure increases for 1 of these QTLs. These results confirm the existence of blood pressure QTLs on rat chromosome 2 and demonstrate the applicability of a speed congenic strategy in the rat and emphasize the important role of the genetic background.

  12. Genetic dissection of milk yield traits and mastitis resistance quantitative trait loci on chromosome 20 in dairy cattle.

    PubMed

    Kadri, Naveen K; Guldbrandtsen, Bernt; Lund, Mogens S; Sahana, Goutam

    2015-12-01

    Intense selection to increase milk yield has had negative consequences for mastitis incidence in dairy cattle. Due to low heritability of mastitis resistance and an unfavorable genetic correlation with milk yield, a reduction in mastitis through traditional breeding has been difficult to achieve. Here, we examined quantitative trait loci (QTL) that segregate for clinical mastitis and milk yield on Bos taurus autosome 20 (BTA20) to determine whether both traits are affected by a single polymorphism (pleiotropy) or by multiple closely linked polymorphisms. In the latter but not the former situation, undesirable genetic correlation could potentially be broken by selecting animals that have favorable variants for both traits. First, we performed a within-breed association study using a haplotype-based method in Danish Holstein cattle (HOL). Next, we analyzed Nordic Red dairy cattle (RDC) and Danish Jersey cattle (JER) with the goal of determining whether these QTL identified in Holsteins were segregating across breeds. Genotypes for 12,566 animals (5,966 HOL, 5,458 RDC, and 1,142 JER) were determined by using the Illumina Bovine SNP50 BeadChip (50K; Illumina, San Diego, CA), which identifies 1,568 single nucleotide polymorphisms on BTA20. Data were combined, phased, and clustered into haplotype states, followed by within- and across-breed haplotype-based association analyses using a linear mixed model. Association signals for both clinical mastitis and milk yield peaked in the 26- to 40-Mb region on BTA20 in HOL. Single-variant association analyses were carried out in the QTL region using whole sequence level variants imputed from references of 2,036 HD genotypes (BovineHD BeadChip; Illumina) and 242 whole-genome sequences. The milk QTL were also segregating in RDC and JER on the BTA20-targeted region; however, an indication of differences in the causal factor(s) was observed across breeds. A previously reported F279Y mutation (rs385640152) within the growth hormone

  13. Polyomino Dissections

    ERIC Educational Resources Information Center

    Hohn, Tiina; Liu, Andy

    2012-01-01

    One of Gardner's passions was to introduce puzzles into the classroom. From this point of view, polyomino dissections are an excellent topic. They require little background, provide training in geometric visualization, and mostly they are fun. In this article, we put together a large collection of such puzzles, introduce a new approach in solving…

  14. Cross-Species PTM Mapping from Phosphoproteomic Data.

    PubMed

    Chaudhuri, Rima; Yang, Jean Yee Hwa

    2017-01-01

    Protein post-translational modifications (PTMs) are crucial for signal transduction in cells. In order to understand key cell signaling events, identification of functionally important PTMs, which are more likely to be evolutionarily conserved, is necessary. In recent times, high-throughput mass spectrometry (MS) has made quantitative datasets in diverse species readily available, which has led to a growing need for tools to facilitate cross-species comparison of PTM data. Cross-species comparison of PTM sites is difficult since they often lie in structurally disordered protein domains. Current tools that address this can only map known PTMs between species based on previously annotated orthologous phosphosites and do not enable cross-species mapping of newly identified modification sites. Here, we describe an automated web-based tool, PhosphOrtholog, that accurately maps annotated and novel orthologous PTM sites from high-throughput MS-based experimental data obtained from different species without relying on existing PTM databases. Identification of conserved PTMs across species from large-scale experimental data increases our knowledgebase of evolutionarily conserved and functional PTM sites that influence most biological processes. In this Chapter, we illustrate with examples how to use PhosphOrtholog to map novel PTM sites from cross-species MS-based phosphoproteomics data.

  15. TiSH--a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC.

    PubMed

    Engholm-Keller, Kasper; Birck, Pernille; Størling, Joachim; Pociot, Flemming; Mandrup-Poulsen, Thomas; Larsen, Martin R

    2012-10-22

    Large scale quantitative phosphoproteomics depends upon multidimensional strategies for peptide fractionation, phosphopeptide enrichment, and mass spectrometric analysis. Previously, most robust comprehensive large-scale phosphoproteomics strategies have relied on milligram amounts of protein. We have set up a multi-dimensional phosphoproteomics strategy combining a number of well-established enrichment and fraction methods: An initial TiO(2) phosphopeptide pre-enrichment step is followed by post-fractionation using sequential elution from IMAC (SIMAC) to separate multi- and mono-phosphorylated peptides, and hydrophilic interaction liquid chromatography (HILIC) of the mono-phosphorylated peptides (collectively abbreviated "TiSH"). The advantages of the strategy include a high specificity and sample preparation workload reduction due to the TiO(2) pre-enrichment step, as well as low adsorptive losses. We demonstrate the capability of this strategy by quantitative investigation of early interferon-γ signaling in low quantities of insulinoma cells. We identified ~6600 unique phosphopeptides from 300 μg of peptides/condition (22 unique phosphopeptides/μg) in a duplex dimethyl labeling experiment, with an enrichment specificity>94%. When doing network analysis of putative phosphorylation changes it could be noted that the identified protein interaction network centered upon proteins known to be affected by the interferon-γ pathway, thereby supporting the utility of this global phosphoproteomics strategy. This strategy thus shows great potential for interrogating signaling networks from low amounts of sample with high sensitivity and specificity.

  16. Phosphoproteomics reveals ALK promote cell progress via RAS/JNK pathway in neuroblastoma

    PubMed Central

    Xu, Guofeng; Zhang, Min; Wu, Yeming; Wu, Zhixiang

    2016-01-01

    Emerging evidence suggests receptor tyrosine kinase ALK as a promising therapeutic target in neuroblastoma. However, clinical trials reveal that a limited proportion of ALK-positive neuroblastoma patients experience clinical benefits from Crizotinib, a clinically approved specific inhibitor of ALK. The precise molecular mechanisms of aberrant ALK activity in neuroblastoma remain elusive, limiting the clinical application of ALK as a therapeutic target in neuroblastoma. Here, we describe a deep quantitative phosphoproteomic approach in which Crizotinib-treated neuroblastoma cell lines bearing aberrant ALK are used to investigate downstream regulated phosphoproteins. We identified more than 19,500—and quantitatively analyzed approximately 10,000—phosphorylation sites from each cell line, ultimately detecting 450–790 significantly-regulated phosphorylation sites. Multiple layers of bioinformatic analysis of the significantly-regulated phosphoproteins identified RAS/JNK as a downstream signaling pathway of ALK, independent of the ALK variant present. Further experiments demonstrated that ALK/JNK signaling could be inactivated by either ALK- or JNK-specific inhibitors, resulting in cell growth inhibition by induction of cell cycle arrest and cell apoptosis. Our study broadly defines the phosphoproteome in response to ALK inhibition and provides a resource for further clinical investigation of ALK as therapeutic target for the treatment of neuroblastoma. PMID:27732954

  17. Phosphoproteomic analysis of the response of maize leaves to drought, heat and their combination stress.

    PubMed

    Hu, Xiuli; Wu, Liuji; Zhao, Feiyun; Zhang, Dayong; Li, Nana; Zhu, Guohui; Li, Chaohao; Wang, Wei

    2015-01-01

    Drought and heat stress, especially their combination, greatly affect crop production. Many studies have described transcriptome, proteome and phosphoproteome changes in response of plants to drought or heat stress. However, the study about the phosphoproteomic changes in response of crops to the combination stress is scare. To understand the mechanism of maize responses to the drought and heat combination stress, phosphoproteomic analysis was performed on maize leaves by using multiplex iTRAQ-based quantitative proteomic and LC-MS/MS methods. Five-leaf-stage maize was subjected to drought, heat or their combination, and the leaves were collected. Globally, heat, drought and the combined stress significantly changed the phosphorylation levels of 172, 149, and 144 phosphopeptides, respectively. These phosphopeptides corresponded to 282 proteins. Among them, 23 only responded to the combined stress and could not be predicted from their responses to single stressors; 30 and 75 only responded to drought and heat, respectively. Notably, 19 proteins were phosphorylated on different sites in response to the single and combination stresses. Of the seven significantly enriched phosphorylation motifs identified, two were common for all stresses, two were common for heat and the combined stress, and one was specific to the combined stress. The signaling pathways in which the phosphoproteins were involved clearly differed among the three stresses. Functional characterization of the phosphoproteins and the pathways identified here could lead to new targets for the enhancement of crop stress tolerance, which will be particularly important in the face of climate change and the increasing prevalence of abiotic stressors.

  18. Phosphoproteomic analysis of Her2/neu signaling and inhibition

    PubMed Central

    Bose, Ron; Molina, Henrik; Patterson, A. Scott; Bitok, John K.; Periaswamy, Balamurugan; Bader, Joel S.; Pandey, Akhilesh; Cole, Philip A.

    2006-01-01

    Her2/neu (Her2) is a tyrosine kinase belonging to the EGF receptor (EGFR)/ErbB family and is overexpressed in 20–30% of human breast cancers. We sought to characterize Her2 signal transduction pathways further by using MS-based quantitative proteomics. Stably transfected cell lines overexpressing Her2 or empty vector were generated, and the effect of an EGFR and Her2 selective tyrosine kinase inhibitor, PD168393, on these cells was characterized. Quantitative measurements were obtained on 462 proteins by using the SILAC (stable isotope labeling with amino acids in cell culture) method to monitor three conditions simultaneously. Of these proteins, 198 showed a significant increase in tyrosine phosphorylation in Her2-overexpressing cells, and 81 showed a significant decrease in phosphorylation. Treatment of Her2-overexpressing cells with PD168393 showed rapid reversibility of the majority of the Her2-triggered phosphorylation events. Phosphoproteins that were identified included many known Her2 signaling molecules as well as known EGFR signaling proteins that had not been previously linked to Her2, such as Stat1, Dok1, and δ-catenin. Importantly, several previously uncharacterized Her2 signaling proteins were identified, including Axl tyrosine kinase, the adaptor protein Fyb, and the calcium-binding protein Pdcd-6/Alg-2. We also identified a phosphorylation site in Her2, Y877, which is located in the activation loop of the kinase domain, is distinct from the known C-terminal tail autophosphorylation sites, and may have important implications for regulation of Her2 signaling. Network modeling, which combined phosphoproteomic results with literature-curated protein–protein interaction data, was used to suggest roles for some of the previously unidentified Her2 signaling proteins. PMID:16785428

  19. Dissection of a Quantitative Trait Locus for PR Interval Duration Identifies Tnni3k as a Novel Modulator of Cardiac Conduction

    PubMed Central

    Milano, Annalisa; Sun, Albert Y.; Tang, Hao; Remme, Carol Ann; Moerland, Perry D.; Tanck, Michael W. T.; Pitt, Geoffrey S.; Marchuk, Douglas A.; Bezzina, Connie R.

    2012-01-01

    Atrio-ventricular conduction disease is a common feature in Mendelian rhythm disorders associated with sudden cardiac death and is characterized by prolongation of the PR interval on the surface electrocardiogram (ECG). Prolongation of the PR interval is also a strong predictor of atrial fibrillation, the most prevalent sustained cardiac arrhythmia. Despite the significant genetic component in PR duration variability, the genes regulating PR interval duration remain largely elusive. We here aimed to dissect the quantitative trait locus (QTL) for PR interval duration that we previously mapped in murine F2 progeny of a sensitized 129P2 and FVBN/J cross. To determine the underlying gene responsible for this QTL, genome-wide transcriptional profiling was carried out on myocardial tissue from 109 F2 mice. Expression QTLs (eQTLs) were mapped and the PR interval QTL was inspected for the co-incidence of eQTLs. We further determined the correlation of each of these transcripts to the PR interval. Tnni3k was the only eQTL, mapping to the PR-QTL, with an established abundant cardiac-specific expression pattern and a significant correlation to PR interval duration. Genotype inspection in various inbred mouse strains revealed the presence of at least three independent haplotypes at the Tnni3k locus. Measurement of PR interval duration and Tnni3k mRNA expression levels in six inbred lines identified a positive correlation between the level of Tnni3k mRNA and PR interval duration. Furthermore, in DBA/2J mice overexpressing hTNNI3K, and in DBA.AKR.hrtfm2 congenic mice, which harbor the AKR/J “high-Tnni3k expression” haplotype in the DBA/2J genetic background, PR interval duration was prolonged as compared to DBA/2J wild-type mice (“low-Tnni3k expression” haplotype). Our data provide the first evidence for a role of Tnni3k in controlling the electrocardiographic PR interval indicating a function of Tnni3k in atrio-ventricular conduction. PMID:23236294

  20. [Dissecting emphysema].

    PubMed

    Gurtner, B

    1991-10-29

    We report six observations of pneumomediastinum, due to dental extraction and use of high speed air turbine drill, aspiration of a nut with air trapping, labor in delivery, status asthmaticus, mechanical ventilation, and rectal perforation. Some patients showed widespread extension of dissecting air presenting as subcutaneous emphysema, pneumopericardium or pneumoretroperitoneum. The mediastinum is thought to be a central pump, the diaphragm and the lung acting like bellows, which distribute interstitial air from and into communicating layers on both sides of the diaphragm. Pneumomediastinum does not prove an air leak in the thoracic cage nor does pneumoretroperitoneum absolutely indicate bowel rupture. Review of the literature.

  1. Biology teachers' dissection practices and the influences that lead to their adoption: An exploratory research

    NASA Astrophysics Data System (ADS)

    Milano, Regina Nicole

    The lack of resolution in the on-going animal dissection debate inspired this mixed methods study to identify Connecticut secondary biology teachers' dissection practices and the influences that lead to their adoption. Qualitative findings indicate past experiences, managing objections to dissection, school culture, goals of biology teaching and ethics as major influences on dissection practices with 58.4% (n=7) of the sample dissecting and 41.6% not dissecting (n=5). Quantitative findings reveal gender, standards and curriculum, advantages of dissection and experiences as a student as major influences on dissection practices with 71.9% (n=92) of the sample dissecting and 28.1% (n=36) not dissecting. The study concludes that dissection policies are necessary and imminent in Connecticut school districts. Furthermore, it advises teacher-initiated, qualitative and quantitative assessments to expose disparities between student dissection perspectives and their own, prior to conducting dissection. Finally, it provides suggestions for addressing potential differences including administrative involvement.

  2. Quantitative phosphoproteome analysis of embryonic stem cell differentiation toward blood

    PubMed Central

    Piazzi, Manuela; Williamson, Andrew; Lee, Chia-Fang; Pearson, Stella; Lacaud, Georges; Kouskoff, Valerie; McCubrey, James A.; Cocco, Lucio; Whetton, Anthony D.

    2015-01-01

    Murine embryonic stem (ES) cells can differentiate in vitro into three germ layers (endodermic, mesodermic, ectodermic). Studies on the differentiation of these cells to specific early differentiation stages has been aided by an ES cell line carrying the Green Fluorescent Protein (GFP) targeted to the Brachyury (Bry) locus which marks mesoderm commitment. Furthermore, expression of the Vascular Endothelial Growth Factor receptor 2 (Flk1) along with Bry defines hemangioblast commitment. Isobaric-tag for relative and absolute quantification (iTRAQTM) and phosphopeptide enrichment coupled to liquid chromatography separation and mass spectrometry allow the study of phosphorylation changes occurring at different stages of ES cell development using Bry and Flk1 expression respectively. We identified and relatively quantified 37 phosphoentities which are modulated during mesoderm-induced ES cells differentiation, comparing epiblast-like, early mesoderm and hemangioblast-enriched cells. Among the proteins differentially phosphorylated toward mesoderm differentiation were: the epigenetic regulator Dnmt3b, the protein kinase GSK3b, the chromatin remodeling factor Smarcc1, the transcription factor Utf1; as well as protein specifically related to stem cell differentiation, as Eomes, Hmga2, Ints1 and Rif1. As most key factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding the post-translational modifications driving their regulation during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia. PMID:25890499

  3. Spontaneous Coronary Artery Dissection

    MedlinePlus

    Spontaneous coronary artery dissection (SCAD) Overview By Mayo Clinic Staff Spontaneous coronary artery dissection — sometimes referred to as SCAD — is an ... the blood vessels in the heart. Spontaneous coronary artery dissection (SCAD) can slow or block blood flow ...

  4. Phosphoproteomic profiling of mouse primary HSPCs reveals new regulators of HSPC mobilization

    PubMed Central

    Ficarro, Scott B.; Hutchinson, John N.; Csepanyi-Komi, Roland; Nguyen, Phi T.; Wisniewski, Eva; Sullivan, Jessica; Hofmann, Oliver; Ligeti, Erzsebet; Marto, Jarrod A.; Wagers, Amy J.

    2016-01-01

    Protein phosphorylation is a central mechanism of signal transduction that both positively and negatively regulates protein function. Large-scale studies of the dynamic phosphorylation states of cell signaling systems have been applied extensively in cell lines and whole tissues to reveal critical regulatory networks, and candidate-based evaluations of phosphorylation in rare cell populations have also been informative. However, application of comprehensive profiling technologies to adult stem cell and progenitor populations has been challenging, due in large part to the scarcity of such cells in adult tissues. Here, we combine multicolor flow cytometry with highly efficient 3-dimensional high performance liquid chromatography/mass spectrometry to enable quantitative phosphoproteomic analysis from 200 000 highly purified primary mouse hematopoietic stem and progenitor cells (HSPCs). Using this platform, we identify ARHGAP25 as a novel regulator of HSPC mobilization and demonstrate that ARHGAP25 phosphorylation at serine 363 is an important modulator of its function. Our approach provides a robust platform for large-scale phosphoproteomic analyses performed with limited numbers of rare progenitor cells. Data from our study comprises a new resource for understanding the molecular signaling networks that underlie hematopoietic stem cell mobilization. PMID:27365422

  5. Deep Phosphoproteomic Measurements Pinpointing Drug Induced Protective Mechanisms in Neuronal Cells

    PubMed Central

    Yu, Chengli; Gao, Jing; Zhou, Yanting; Chen, Xiangling; Xiao, Ruoxuan; Zheng, Jing; Liu, Yansheng; Zhou, Hu

    2016-01-01

    Alzheimer's disease (AD) is a progressive and irreversible neurological disorder that impairs the living quality of old population and even life spans. New compounds have shown potential inneuroprotective effects in AD, such as GFKP-19, a 2-pyrrolidone derivative which has been proved to enhance the memory of dysmnesia mouse. The molecular mechanisms remain to be established for these drug candidates. Large-scale phosphoproteomic approach has been evolved rapidly in the last several years, which holds the potential to provide a useful toolkit to understand cellular signaling underlying drug effects. To establish and test such a method, we accurately analyzed the deep quantitative phosphoproteome of the neuro-2a cells treated with and without GFKP-19 using triple SILAC labeling. A total of 14,761 Class I phosphosites were quantified between controls, damaged, and protected conditions using the high resolution mass spectrometry, with a decent inter-mass spectrometer reproducibility for even subtle regulatory events. Our data suggests that GFKP-19 can reverse Aβ25−35 induced phosphorylation change in neuro-2a cells, and might protect the neuron system in two ways: firstly, it may decrease oxidative damage and inflammation induced by NO via down regulating the phosphorylation of nitric oxide synthase NOS1 at S847; Secondly, it may decrease tau protein phosphorylation through down-regulating the phosphorylation level of MAPK14 at T180. All mass spectrometry data are available via ProteomeXchange with identifier PXD005312. PMID:28066266

  6. Phosphoproteomic analysis reveals compensatory effects in the piriform cortex of VX nerve agent exposed rats.

    PubMed

    Nirujogi, Raja Sekhar; Wright, James D; Manda, Srikanth S; Zhong, Jun; Na, Chan Hyun; Meyerhoff, James; Benton, Bernard; Jabbour, Rabih; Willis, Kristen; Kim, Min-Sik; Pandey, Akhilesh; Sekowski, Jennifer W

    2015-01-01

    To gain insights into the toxicity induced by the nerve agent VX, an MS-based phosphoproteomic analysis was carried out on the piriform cortex region of brains from VX-treated rats. Using isobaric tag based TMT labeling followed by titanium dioxide enrichment strategy, we identified 9975 unique phosphosites derived from 3287 phosphoproteins. Temporal changes in the phosphorylation status of peptides were observed over a time period of 24 h in rats exposed to a 1× LD50, intravenous (i.v.) dose with the most notable changes occurring at the 1 h postexposure time point. Five major functional classes of proteins exhibited changes in their phosphorylation status: (i) ion channels/transporters, including ATPases, (ii) kinases/phosphatases, (iii) GTPases, (iv) structural proteins, and (v) transcriptional regulatory proteins. This study is the first quantitative phosphoproteomic analysis of VX toxicity in the brain. Understanding the toxicity and compensatory signaling mechanisms will improve the understanding of the complex toxicity of VX in the brain and aid in the elucidation of novel molecular targets that would be important for development of improved countermeasures. All MS data have been deposited in the ProteomeXchange with identifier PXD001184 (http://proteomecentral.proteomexchange.org/dataset/PXD001184).

  7. Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants

    PubMed Central

    Greenwood, Edward JD; Matheson, Nicholas J; Wals, Kim; van den Boomen, Dick JH; Antrobus, Robin; Williamson, James C; Lehner, Paul J

    2016-01-01

    Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function. DOI: http://dx.doi.org/10.7554/eLife.18296.001 PMID:27690223

  8. Shotguns in the front line: phosphoproteomics in plants.

    PubMed

    Nakagami, Hirofumi; Sugiyama, Naoyuki; Ishihama, Yasushi; Shirasu, Ken

    2012-01-01

    The emergence of 'shotgun proteomics' has paved the way for high-throughput proteome analysis, by which thousands of proteins can be identified simultaneously from complex samples. Although the shotgun approach has the potential to monitor many different post-translational modifications, further technological development is needed to enrich each post-translational 'modificome'. Large-scale in vivo phosphorylation site mapping, so-called shotgun phosphoproteomics, has become feasible in various organisms, including plants, owing to recent technological breakthroughs. Shotgun phosphoproteomics is not a mature technology, but progress has been rapid. In this review, we highlight the scope and limitations of current methods, and some key technological issues in this field.

  9. Versatile nanocomposites in phosphoproteomics: a review.

    PubMed

    Najam-ul-Haq, Muhammad; Jabeen, Fahmida; Hussain, Dilshad; Saeed, Adeela; Musharraf, Syed Ghulam; Huck, Christian W; Bonn, Günther K

    2012-10-17

    Protein phosphorylation is one of the most important post-translational modifications. Phosphorylated peptides are present in low abundance in blood serum but play a vital role in regulatory mechanisms and may serve as casual factors in diseases. The enrichment and analysis of phosphorylated peptides directly from human serum and mapping the phosphorylation sites is a challenging task. Versatile nanocomposites of different materials have been synthesized using simple but efficient methodologies for their enrichment. The nanocomposites include magnetic, coated, embedded as well as chemically derivatized materials. Different base materials such as polymers, carbon based and metal oxides are used. The comparison of nanocomposites with respective nanoparticles provides sufficient facts about their efficiency in terms of loading capacity and capture efficiency. The cost for preparing them is low and they hold great promise to be used as chromatographic materials for phosphopeptide enrichment. This review gives an overview of different nanocomposites in phosphoproteomics, discussing the improved efficiency than the individual counterparts and highlighting their significance in phosphopeptide enrichment.

  10. Phosphoproteomics by mass spectrometry and classical protein chemistry approaches.

    PubMed

    Salih, Erdjan

    2005-01-01

    spectrometry have clearly revolutionized the studies of phosphoprotein biochemistry, and include the development of specific strategies to preferentially enrich phosphoproteins by covalent-modifications that incorporate affinity tags that use the physicochemical properties of phosphoaminoacids. The phosphoserine/phosphothreonine-containing proteins/peptides are derivatized under base-catalyzed conditions by thiol agents; mono- and di-thiol reagents both have been used in such studies. The thiol agent may have: (i) an affinity tag for protein enrichment; (ii) stable-isotopic variants for relative quantitation; or (iii) a combination of the moieties in (i) and (ii). These strategies and techniques, together with others, are reviewed, including their practical application to the study of phosphoprotein biochemistry and structure-function. The consensus of how classical protein chemistry and current MS technology overlap into special case of proteomics, namely "phosphoproteomics," will be discussed.

  11. Hippocampal phosphoproteomics of F344 rats exposed to 1-bromopropane

    SciTech Connect

    Huang, Zhenlie; Ichihara, Sahoko; Oikawa, Shinji; Chang, Jie; Zhang, Lingyi; Hu, Shijie; Huang, Hanlin; Ichihara, Gaku

    2015-01-15

    1-Bromopropane (1-BP) is neurotoxic in both experimental animals and human. To identify phosphorylated modification on the unrecognized post-translational modifications of proteins and investigate their role in 1-BP-induced neurotoxicity, changes in hippocampal phosphoprotein expression levels were analyzed quantitatively in male F344 rats exposed to 1-BP inhalation at 0, 400, or 1000 ppm for 8 h/day for 1 or 4 weeks. Hippocampal protein extracts were analyzed qualitatively and quantitatively by Pro-Q Diamond gel staining and SYPRO Ruby staining coupled with two-dimensional difference in gel electrophoresis (2D-DIGE), respectively, as well as by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to identify phosphoproteins. Changes in selected proteins were further confirmed by Manganese II (Mn{sup 2+})-Phos-tag SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Bax and cytochrome c protein levels were determined by western blotting. Pro-Q Diamond gel staining combined with 2D-DIGE identified 26 phosphoprotein spots (p < 0.05), and MALDI-TOF/MS identified 18 up-regulated proteins and 8 down-regulated proteins. These proteins are involved in the biological process of response to stimuli, metabolic processes, and apoptosis signaling. Changes in the expression of phosphorylated 14-3-3 θ were further confirmed by Mn{sup 2+}-Phos-tag SDS-PAGE. Western blotting showed overexpression of Bax protein in the mitochondria with down-regulation in the cytoplasm, whereas cytochrome c expression was high in the cytoplasm but low in the mitochondria after 1-BP exposure. Our results suggest that the pathogenesis of 1-BP-induced hippocampal damage involves inhibition of antiapoptosis process. Phosphoproteins identified in this study can potentially serve as biomarkers for 1-BP-induced neurotoxicity. - Highlights: • 1-BP modified hippocampal phosphoproteome in rat and 23 altered proteins were identified. • 1-BP changed phosphorylation

  12. Spatial proteomic and phospho-proteomic organization in three prototypical cell migration modes

    PubMed Central

    2014-01-01

    Background Tight spatio-temporal signaling of cytoskeletal and adhesion dynamics is required for localized membrane protrusion that drives directed cell migration. Different ensembles of proteins are therefore likely to get recruited and phosphorylated in membrane protrusions in response to specific cues. Results Here, we use an assay that allows to biochemically purify extending protrusions of cells migrating in response to three prototypical receptors: integrins, recepor tyrosine kinases and G-coupled protein receptors. Using quantitative proteomics and phospho-proteomics approaches, we provide evidence for the existence of cue-specific, spatially distinct protein networks in the different cell migration modes. Conclusions The integrated analysis of the large-scale experimental data with protein information from databases allows us to understand some emergent properties of spatial regulation of signaling during cell migration. This provides the cell migration community with a large-scale view of the distribution of proteins and phospho-proteins regulating directed cell migration. PMID:24987309

  13. Battle through Signaling between Wheat and the Fungal Pathogen Septoria tritici Revealed by Proteomics and Phosphoproteomics*

    PubMed Central

    Yang, Fen; Melo-Braga, Marcella N.; Larsen, Martin R.; Jørgensen, Hans J. L.; Palmisano, Giuseppe

    2013-01-01

    The fungus Septoria tritici causes the disease septoria tritici blotch in wheat, one of the most economically devastating foliar diseases in this crop. To investigate signaling events and defense responses in the wheat–S. tritici interaction, we performed a time-course study of S. tritici infection in resistant and susceptible wheat using quantitative proteomics and phosphoproteomics, with special emphasis on the initial biotrophic phase of interactions. Our study revealed an accumulation of defense and stress-related proteins, suppression of photosynthesis, and changes in sugar metabolism during compatible and incompatible interactions. However, differential regulation of the phosphorylation status of signaling proteins, transcription and translation regulators, and membrane-associated proteins was observed between two interactions. The proteomic data were correlated with a more rapid or stronger accumulation of signal molecules, including calcium, H2O2, NO, and sugars, in the resistant than in the susceptible cultivar in response to the infection. Additionally, 31 proteins and 5 phosphoproteins from the pathogen were identified, including metabolic proteins and signaling proteins such as GTP-binding proteins, 14–3-3 proteins, and calcium-binding proteins. Quantitative PCR analysis showed the expression of fungal signaling genes and genes encoding a superoxide dismutase and cell-wall degrading enzymes. These results indicate roles of signaling, antioxidative stress mechanisms, and nutrient acquisition in facilitating the initial symptomless growth. Taken in its entirety, our dataset suggests interplay between the plant and S. tritici through complex signaling networks and downstream molecular events. Resistance is likely related to several rapidly and intensively triggered signal transduction cascades resulting in a multiple-level activation of transcription and translation processes of defense responses. Our sensitive approaches and model provide a

  14. Phosphoproteome Profiling Reveals Circadian Clock Regulation of Posttranslational Modifications in the Murine Hippocampus.

    PubMed

    Chiang, Cheng-Kang; Xu, Bo; Mehta, Neel; Mayne, Janice; Sun, Warren Y L; Cheng, Kai; Ning, Zhibin; Dong, Jing; Zou, Hanfa; Cheng, Hai-Ying Mary; Figeys, Daniel

    2017-01-01

    The circadian clock is an endogenous oscillator that drives daily rhythms in physiology, behavior, and gene expression. The underlying mechanisms of circadian timekeeping are cell-autonomous and involve oscillatory expression of core clock genes that is driven by interconnecting transcription-translation feedback loops (TTFLs). Circadian clock TTFLs are further regulated by posttranslational modifications, in particular, phosphorylation. The hippocampus plays an important role in spatial memory and the conversion of short- to long-term memory. Several studies have reported the presence of a peripheral oscillator in the hippocampus and have highlighted the importance of circadian regulation in memory formation. Given the general importance of phosphorylation in circadian clock regulation, we performed global quantitative proteome and phosphoproteome analyses of the murine hippocampus across the circadian cycle, applying spiked-in labeled reference and high accuracy mass spectrometry (MS). Of the 3,052 proteins and 2,868 phosphosites on 1,368 proteins that were accurately quantified, 1.7% of proteins and 5.2% of phosphorylation events exhibited time-of-day-dependent expression profiles. The majority of circadian phosphopeptides displayed abrupt fluctuations at mid-to-late day without underlying rhythms of protein abundance. Bioinformatic analysis of cyclic phosphorylation events revealed their diverse distribution in different biological pathways, most notably, cytoskeletal organization and neuronal morphogenesis. This study provides the first large-scale, quantitative MS analysis of the circadian phosphoproteome and proteome of the murine hippocampus and highlights the significance of rhythmic regulation at the posttranslational level in this peripheral oscillator. In addition to providing molecular insights into the hippocampal circadian clock, our results will assist in the understanding of genetic factors that underlie rhythms-associated pathological states of

  15. Phosphoproteome Profiling Reveals Circadian Clock Regulation of Posttranslational Modifications in the Murine Hippocampus

    PubMed Central

    Chiang, Cheng-Kang; Xu, Bo; Mehta, Neel; Mayne, Janice; Sun, Warren Y. L.; Cheng, Kai; Ning, Zhibin; Dong, Jing; Zou, Hanfa; Cheng, Hai-Ying Mary; Figeys, Daniel

    2017-01-01

    The circadian clock is an endogenous oscillator that drives daily rhythms in physiology, behavior, and gene expression. The underlying mechanisms of circadian timekeeping are cell-autonomous and involve oscillatory expression of core clock genes that is driven by interconnecting transcription–translation feedback loops (TTFLs). Circadian clock TTFLs are further regulated by posttranslational modifications, in particular, phosphorylation. The hippocampus plays an important role in spatial memory and the conversion of short- to long-term memory. Several studies have reported the presence of a peripheral oscillator in the hippocampus and have highlighted the importance of circadian regulation in memory formation. Given the general importance of phosphorylation in circadian clock regulation, we performed global quantitative proteome and phosphoproteome analyses of the murine hippocampus across the circadian cycle, applying spiked-in labeled reference and high accuracy mass spectrometry (MS). Of the 3,052 proteins and 2,868 phosphosites on 1,368 proteins that were accurately quantified, 1.7% of proteins and 5.2% of phosphorylation events exhibited time-of-day-dependent expression profiles. The majority of circadian phosphopeptides displayed abrupt fluctuations at mid-to-late day without underlying rhythms of protein abundance. Bioinformatic analysis of cyclic phosphorylation events revealed their diverse distribution in different biological pathways, most notably, cytoskeletal organization and neuronal morphogenesis. This study provides the first large-scale, quantitative MS analysis of the circadian phosphoproteome and proteome of the murine hippocampus and highlights the significance of rhythmic regulation at the posttranslational level in this peripheral oscillator. In addition to providing molecular insights into the hippocampal circadian clock, our results will assist in the understanding of genetic factors that underlie rhythms-associated pathological states of

  16. Dissecting Classroom Ethics.

    ERIC Educational Resources Information Center

    Allchin, Douglas

    1991-01-01

    Described are activities that lead to values clarification. Issues such as dissection, bioengineering, birth control, medical resources, and death are discussed. Included is a student questionnaire on the subject of dissection and the use of animals in laboratories. (KR)

  17. Data set from a comprehensive phosphoproteomic analysis of rice variety IRBB5 in response to bacterial blight.

    PubMed

    Hou, Yuxuan; Tong, Xiaohong; Wang, Yifeng; Qiu, Jiehua; Li, Zhiyong; Zhang, Wen; Huang, Shiwen; Zhang, Jian

    2016-03-01

    Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) has become one of the most devastating diseases for rice, a major food source for over half of the world populations. To investigate the roles of protein phosphorylation in rice bacterial blight resistance, a quantitative phosphoproteomic study was conducted in rice variety IRBB5 at 0 h and 24 h after Xoo infection. 2367 and 2223 phosphosites on 1334 and 1297 representative proteins were identified in 0 h and 24 h after Xoo infection, respectively, out of which 762 proteins were found to be differentially phosphorylated. In associated with the published article "A comprehensive quantitative phosphoproteome analysis of rice in response to bacterial blight" in BMC Plant Biology (Hou et al., 2015) [1], this dataset article provided the detailed information of experimental designing, methods, features as well as the raw data of mass spectrometry (MS) identification. The MS proteomics data could be fully accessed from the ProteomeXchange Consortium with the dataset identifier PXD002222.

  18. Proteomics and Phosphoproteomics Analysis of Human Lens Fiber Cell Membranes

    PubMed Central

    Wang, Zhen; Han, Jun; David, Larry L.; Schey, Kevin L.

    2013-01-01

    Purpose. The human lens fiber cell insoluble membrane fraction contains important membrane proteins, cytoskeletal proteins, and cytosolic proteins that are strongly associated with the membrane. The purpose of this study was to characterize the lens fiber cell membrane proteome and phosphoproteome from human lenses. Methods. HPLC-mass spectrometry–based multidimensional protein identification technology (MudPIT), without or with phosphopeptide enrichment, was applied to study the proteome and phosphoproteome of lens fiber cell membranes, respectively. Results. In total, 951 proteins were identified, including 379 integral membrane and membrane-associated proteins. Enriched gene categories and pathways based on the proteomic analysis include carbohydrate metabolism (glycolysis/gluconeogenesis, pentose phosphate pathway, pyruvate metabolism), proteasome, cell-cell signaling and communication (GTP binding, gap junction, focal adhesion), glutathione metabolism, and actin regulation. The combination of TiO2 phosphopeptide enrichment and MudPIT analysis revealed 855 phosphorylation sites on 271 proteins, including 455 phosphorylation sites that have not been previously identified. PKA, PKC, CKII, p38MAPK, and RSK are predicted as the major kinases for phosphorylation on the sites identified in the human lens membrane fraction. Conclusions. The results presented herein significantly expand the characterized proteome and phosphoproteome of the human lens fiber cell and provide a valuable reference for future research in studies of lens development and disease. PMID:23349431

  19. The Problems of Dissection.

    ERIC Educational Resources Information Center

    Davis, Pat

    1997-01-01

    Describes some problems of classroom dissection including the cruelty that animals destined for the laboratory suffer. Discusses the multilevel approach that the National Anti-Vivisection Society (NAVS) has developed to address the problems of animal dissection such as offering a dissection hotline, exhibiting at science teacher conferences, and…

  20. Laparoscopic dissecting instruments.

    PubMed

    Park, A E; Mastrangelo, M J; Gandsas, A; Chu, U; Quick, N E

    2001-03-01

    The authors provide an overview of laparoscopic dissecting instruments and discuss early development, surgical options, and special features. End effectors of different shapes and functions are described. A comparison of available energy sources for laparoscopic instruments includes discussion of thermal dissection, ultrasonic dissection, and water-jet dissection. The ergonomic risks and challenges inherent in the use of current laparoscopic instruments are outlined, as well as ergonomic issues for the design of future instruments. New directions that laparoscopic instrumentation may take are considered in connection with developing technology in robotics, haptic feedback, and MicroElectroMechanical Systems.

  1. Using SNP markers to dissect linkage disequilibrium at a major quantitative trait locus for resistance to the potato cyst nematode Globodera pallida on potato chromosome V.

    PubMed

    Achenbach, Ute; Paulo, Joao; Ilarionova, Evgenyia; Lübeck, Jens; Strahwald, Josef; Tacke, Eckhard; Hofferbert, Hans-Reinhard; Gebhardt, Christiane

    2009-02-01

    The damage caused by the parasitic root cyst nematode Globodera pallida is a major yield-limiting factor in potato cultivation . Breeding for resistance is facilitated by the PCR-based marker 'HC', which is diagnostic for an allele conferring high resistance against G. pallida pathotype Pa2/3 that has been introgressed from the wild potato species Solanum vernei into the Solanum tuberosum tetraploid breeding pool. The major quantitative trait locus (QTL) controlling this nematode resistance maps on potato chromosome V in a hot spot for resistance to various pathogens including nematodes and the oomycete Phytophthora infestans. An unstructured sample of 79 tetraploid, highly heterozygous varieties and breeding clones was selected based on presence (41 genotypes) or absence (38 genotypes) of the HC marker. Testing the clones for resistance to G. pallida confirmed the diagnostic power of the HC marker. The 79 individuals were genotyped for 100 single nucleotide polymorphisms (SNPs) at 10 loci distributed over 38 cM on chromosome V. Forty-five SNPs at six loci spanning 2 cM in the interval between markers GP21-GP179 were associated with resistance to G. pallida. Based on linkage disequilibrium (LD) between SNP markers, six LD groups comprising between 2 and 18 SNPs were identified. The LD groups indicated the existence of multiple alleles at a single resistance locus or at several, physically linked resistance loci. LD group C comprising 18 SNPs corresponded to the 'HC' marker. LD group E included 16 SNPs and showed an association peak, which positioned one nematode resistance locus physically close to the R1 gene family.

  2. Search Databases and Statistics: Pitfalls and Best Practices in Phosphoproteomics.

    PubMed

    Refsgaard, Jan C; Munk, Stephanie; Jensen, Lars J

    2016-01-01

    Advances in mass spectrometric instrumentation in the past 15 years have resulted in an explosion in the raw data yield from typical phosphoproteomics workflows. This poses the challenge of confidently identifying peptide sequences, localizing phosphosites to proteins and quantifying these from the vast amounts of raw data. This task is tackled by computational tools implementing algorithms that match the experimental data to databases, providing the user with lists for downstream analysis. Several platforms for such automated interpretation of mass spectrometric data have been developed, each having strengths and weaknesses that must be considered for the individual needs. These are reviewed in this chapter. Equally critical for generating highly confident output datasets is the application of sound statistical criteria to limit the inclusion of incorrect peptide identifications from database searches. Additionally, careful filtering and use of appropriate statistical tests on the output datasets affects the quality of all downstream analyses and interpretation of the data. Our considerations and general practices on these aspects of phosphoproteomics data processing are presented here.

  3. Phosphoproteome Dynamics Upon Changes in Plant Water Status Reveal Early Events Associated With Rapid Growth Adjustment in Maize Leaves*

    PubMed Central

    Bonhomme, Ludovic; Valot, Benoît; Tardieu, François; Zivy, Michel

    2012-01-01

    Plant growth adjustment during water deficit is a crucial adaptive response. The rapid fine-tuned control achieved at the post-translational level is believed to be of considerable importance for regulating early changes in plant growth reprogramming. Aiming at a better understanding of early responses to contrasting plant water statuses, we carried out a survey of the protein phosphorylation events in the growing zone of maize leaves upon a range of water regimes. In this study, the impact of mild and severe water deficits were evaluated in comparison with constant optimal watering and with recovery periods lasting 5, 10, 20, 30, 45, and 60 min. Using four biological replicates per treatment and a robust quantitative phosphoproteomic methodology based on stable-isotope labeling, we identified 3664 unique phosphorylation sites on 2496 proteins. The abundance of nearly 1250 phosphorylated peptides was reproducibly quantified and profiled with high confidence among treatments. A total of 138 phosphopeptides displayed highly significant changes according to water regimes and enabled to identify specific patterns of response to changing plant water statuses. Further quantification of protein amounts emphasized that most phosphorylation changes did not reflect protein abundance variation. During water deficit and recovery, extensive changes in phosphorylation status occurred in critical regulators directly or indirectly involved in plant growth and development. These included proteins influencing epigenetic control, gene expression, cell cycle-dependent processes and phytohormone-mediated responses. Some of the changes depended on stress intensity whereas others depended on rehydration duration, including rapid recoveries that occurred as early as 5 or 10 mins after rewatering. By combining a physiological approach and a quantitative phosphoproteomic analysis, this work provides new insights into the in vivo early phosphorylation events triggered by rapid changes in

  4. Phosphoproteome dynamics upon changes in plant water status reveal early events associated with rapid growth adjustment in maize leaves.

    PubMed

    Bonhomme, Ludovic; Valot, Benoît; Tardieu, François; Zivy, Michel

    2012-10-01

    Plant growth adjustment during water deficit is a crucial adaptive response. The rapid fine-tuned control achieved at the post-translational level is believed to be of considerable importance for regulating early changes in plant growth reprogramming. Aiming at a better understanding of early responses to contrasting plant water statuses, we carried out a survey of the protein phosphorylation events in the growing zone of maize leaves upon a range of water regimes. In this study, the impact of mild and severe water deficits were evaluated in comparison with constant optimal watering and with recovery periods lasting 5, 10, 20, 30, 45, and 60 min. Using four biological replicates per treatment and a robust quantitative phosphoproteomic methodology based on stable-isotope labeling, we identified 3664 unique phosphorylation sites on 2496 proteins. The abundance of nearly 1250 phosphorylated peptides was reproducibly quantified and profiled with high confidence among treatments. A total of 138 phosphopeptides displayed highly significant changes according to water regimes and enabled to identify specific patterns of response to changing plant water statuses. Further quantification of protein amounts emphasized that most phosphorylation changes did not reflect protein abundance variation. During water deficit and recovery, extensive changes in phosphorylation status occurred in critical regulators directly or indirectly involved in plant growth and development. These included proteins influencing epigenetic control, gene expression, cell cycle-dependent processes and phytohormone-mediated responses. Some of the changes depended on stress intensity whereas others depended on rehydration duration, including rapid recoveries that occurred as early as 5 or 10 mins after rewatering. By combining a physiological approach and a quantitative phosphoproteomic analysis, this work provides new insights into the in vivo early phosphorylation events triggered by rapid changes in

  5. In-depth phosphoproteomic analysis of royal jelly derived from western and eastern honeybee species.

    PubMed

    Han, Bin; Fang, Yu; Feng, Mao; Lu, Xiaoshan; Huo, Xinmei; Meng, Lifeng; Wu, Bin; Li, Jianke

    2014-12-05

    The proteins in royal jelly (RJ) play a pivotal role in the nutrition, immune defense, and cast determination of honeybee larvae and have a wide range of pharmacological and health-promoting functions for humans as well. Although the importance of post-translational modifications (PTMs) in protein function is known, investigation of protein phosphorylation of RJ proteins is still very limited. To this end, two complementary phosphopeptide enrichment materials (Ti(4+)-IMAC and TiO2) and high-sensitivity mass spectrometry were applied to establish a detailed phosphoproteome map and to qualitatively and quantitatively compare the phosphoproteomes of RJ produced by Apis mellifera ligustica (Aml) and Apis cerana cerana (Acc). In total, 16 phosphoproteins carrying 67 phosphorylation sites were identified in RJ derived from western bees, and nine proteins phosphorylated on 71 sites were found in RJ produced by eastern honeybees. Of which, eight phosphorylated proteins were common to both RJ samples, and the same motif ([S-x-E]) was extracted, suggesting that the function of major RJ proteins as nutrients and immune agents is evolutionary preserved in both of these honeybee species. All eight overlapping phosphoproteins showed significantly higher abundance in Acc-RJ than in Aml-RJ, and the phosphorylation of Jelleine-II (an antimicrobial peptide, TPFKLSLHL) at S(6) in Acc-RJ had stronger antimicrobial properties than that at T(1) in Aml-RJ even though the overall antimicrobial activity of Jelleine-II was found to decrease after phosphorylation. The differences in phosphosites, peptide abundance, and antimicrobial activity of the phosphorylated RJ proteins indicate that the two major honeybee species employ distinct phosphorylation strategies that align with their different biological characteristics shaped by evolution. The phosphorylation of RJ proteins are potentially driven by the activity of extracellular serine/threonine protein kinase FAM20C-like protein (FAM20C

  6. Mechanisms of Soybean Roots' Tolerances to Salinity Revealed by Proteomic and Phosphoproteomic Comparisons Between Two Cultivars*

    PubMed Central

    Pi, Erxu; Qu, Liqun; Hu, Jianwen; Huang, Yingying; Qiu, Lijuan; Lu, Hongfei; Jiang, Bo; Liu, Cong; Peng, Tingting; Zhao, Ying; Wang, Huizhong; Tsai, Sau-Na; Ngai, Saiming; Du, Liqun

    2016-01-01

    Understanding molecular mechanisms underlying plant salinity tolerance provides valuable knowledgebase for effective crop improvement through genetic engineering. Current proteomic technologies, which support reliable and high-throughput analyses, have been broadly used for exploring sophisticated molecular networks in plants. In the current study, we compared phosphoproteomic and proteomic changes in roots of different soybean seedlings of a salt-tolerant cultivar (Wenfeng07) and a salt-sensitive cultivar (Union85140) induced by salt stress. The root samples of Wenfeng07 and Union85140 at three-trifoliate stage were collected at 0 h, 0.5 h, 1 h, 4 h, 12 h, 24 h, and 48 h after been treated with 150 mm NaCl. LC-MS/MS based phosphoproteomic analysis of these samples identified a total of 2692 phosphoproteins and 5509 phosphorylation sites. Of these, 2344 phosphoproteins containing 3744 phosphorylation sites were quantitatively analyzed. Our results showed that 1163 phosphorylation sites were differentially phosphorylated in the two compared cultivars. Among them, 10 MYB/MYB transcription factor like proteins were identified with fluctuating phosphorylation modifications at different time points, indicating that their crucial roles in regulating flavonol accumulation might be mediated by phosphorylated modifications. In addition, the protein expression profiles of these two cultivars were compared using LC MS/MS based shotgun proteomic analysis, and expression pattern of all the 89 differentially expressed proteins were independently confirmed by qRT-PCR. Interestingly, the enzymes involved in chalcone metabolic pathway exhibited positive correlations with salt tolerance. We confirmed the functional relevance of chalcone synthase, chalcone isomerase, and cytochrome P450 monooxygenase genes using soybean composites and Arabidopsis thaliana mutants, and found that their salt tolerance were positively regulated by chalcone synthase, but was negatively regulated by

  7. Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro *

    PubMed Central

    Fíla, Jan; Radau, Sonja; Matros, Andrea; Hartmann, Anja; Scholz, Uwe; Feciková, Jana; Mock, Hans-Peter; Čapková, Věra; Zahedi, René Peiman; Honys, David

    2016-01-01

    Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved. PMID:26792808

  8. Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro.

    PubMed

    Fíla, Jan; Radau, Sonja; Matros, Andrea; Hartmann, Anja; Scholz, Uwe; Feciková, Jana; Mock, Hans-Peter; Čapková, Věra; Zahedi, René Peiman; Honys, David

    2016-04-01

    Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved.

  9. Phosphoproteomics Identifies CK2 as a Negative Regulator of Beige Adipocyte Thermogenesis and Energy Expenditure.

    PubMed

    Shinoda, Kosaku; Ohyama, Kana; Hasegawa, Yutaka; Chang, Hsin-Yi; Ogura, Mayu; Sato, Ayaka; Hong, Haemin; Hosono, Takashi; Sharp, Louis Z; Scheel, David W; Graham, Mark; Ishihama, Yasushi; Kajimura, Shingo

    2015-12-01

    Catecholamines promote lipolysis both in brown and white adipocytes, whereas the same stimuli preferentially activate thermogenesis in brown adipocytes. Molecular mechanisms for the adipose-selective activation of thermogenesis remain poorly understood. Here, we employed quantitative phosphoproteomics to map global and temporal phosphorylation profiles in brown, beige, and white adipocytes under β3-adrenenoceptor activation and identified kinases responsible for the adipose-selective phosphorylation profiles. We found that casein kinase2 (CK2) activity is preferentially higher in white adipocytes than brown/beige adipocytes. Genetic or pharmacological blockade of CK2 in white adipocytes activates the thermogenic program in response to cAMP stimuli. Such activation is largely through reduced CK2-mediated phosphorylation of class I HDACs. Notably, inhibition of CK2 promotes beige adipocyte biogenesis and leads to an increase in whole-body energy expenditure and ameliorates diet-induced obesity and insulin resistance. These results indicate that CK2 is a plausible target to rewire the β3-adrenenoceptor signaling cascade that promotes thermogenesis in adipocytes.

  10. Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast.

    PubMed

    Bodenmiller, Bernd; Wanka, Stefanie; Kraft, Claudine; Urban, Jörg; Campbell, David; Pedrioli, Patrick G; Gerrits, Bertran; Picotti, Paola; Lam, Henry; Vitek, Olga; Brusniak, Mi-Youn; Roschitzki, Bernd; Zhang, Chao; Shokat, Kevan M; Schlapbach, Ralph; Colman-Lerner, Alejandro; Nolan, Garry P; Nesvizhskii, Alexey I; Peter, Matthias; Loewith, Robbie; von Mering, Christian; Aebersold, Ruedi

    2010-12-21

    The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases; however, it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as a whole. Here, we used label-free, quantitative phosphoproteomics in a systematically perturbed model organism (Saccharomyces cerevisiae) to determine the relationships between 97 kinases, 27 phosphatases, and more than 1000 phosphoproteins. We identified 8814 regulated phosphorylation events, describing the first system-wide protein phosphorylation network in vivo. Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery-and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis.

  11. GPM Dissects Typhoon Hagupit

    NASA Video Gallery

    NASA/JAXA's GPM Dissects Typhoon Hagupit Animation revealing a swath of NASA/JAXA's Global Precipitation Measurement (GPM) mission's Core Observatory GMI precipitation rates over Typhoon Hagupit. A...

  12. Phosphorylation of proteins during human myometrial contractions: A phosphoproteomic approach.

    PubMed

    Hudson, Claire A; López Bernal, Andrés

    2017-01-22

    Phasic myometrial contractility is a key component of human parturition and the contractions are driven by reversible phosphorylation of myosin light chains catalyzed by the calcium (Ca(2+))-dependent enzyme myosin light chain kinase (MYLK). Other yet unknown phosphorylation or de-phosphorylation events may contribute to myometrial contraction and relaxation. In this study we have performed a global phosphoproteomic analysis of human myometrial tissue using tandem mass tagging to detect changes in the phosphorylation status of individual myometrial proteins during spontaneous and oxytocin-driven phasic contractions. We were able to detect 22 individual phosphopeptides whose relative ratio changed (fold > 2 or < 0.5) in response to spontaneous or oxytocin-stimulated contraction. The most significant changes in phosphorylation were to MYLK on serine 1760, a site associated with reductions in calmodulin binding and subsequent kinase activity. Phosphorylated MYLK (ser1760) increased significantly during spontaneous (9.83 ± 3.27 fold, P < 0.05) and oxytocin -induced (18.56 ± 8.18 fold, P < 0.01) contractions and we were able to validate these data using immunoblotting. Pathway analysis suggested additional proteins involved in calcium signalling, cGMP-PRKG signalling, adrenergic signalling and oxytocin signalling were also phosphorylated during contractions. This study demonstrates that a global phosphoproteomic analysis of myometrial tissue is a sensitive approach to detect changes in the phosphorylation of proteins during myometrial contractions, and provides a platform for further validation of these changes and for identification of their functional significance.

  13. Combining Metabolic ¹⁵N Labeling with Improved Tandem MOAC for Enhanced Probing of the Phosphoproteome.

    PubMed

    Thomas, Martin; Huck, Nicola; Hoehenwarter, Wolfgang; Conrath, Uwe; Beckers, Gerold J M

    2015-01-01

    that is based on the successive enrichment of light and heavy nitrogen-labeled phosphoproteins and peptides. This improved strategy combines metabolic labeling of whole plants with the stable heavy nitrogen isotope ((15)N), protein extraction under denaturing conditions, phosphoprotein enrichment using Al(OH)3-based MOAC, and tryptic digest of enriched phosphoproteins followed by TiO2-based MOAC of phosphopeptides and quantitative phosphopeptide measurement by liquid chromatography (LC) and high-resolution accurate mass (HR/AM) mass spectrometry (MS). Thus, tandem MOAC effectively targets the phosphate moiety of phosphoproteins and phosphopeptides and allows probing of the phosphoproteome to unprecedented depth, while (15)N metabolic labeling enables accurate relative quantification of measured peptides and direct comparison between samples.

  14. Dissection of the interventricular septum

    PubMed Central

    Gu, Xiaoyan; He, Yihua; Luan, Shurong; Zhao, Ying; Sun, Lin; Zhang, Hongjia; Nixon, J.V. Ian

    2017-01-01

    Abstract Dissection of the interventricular septum (IVS) is an extremely rare entity. An institutional echocardiographic database was retrospectively reviewed; 13 patients with a diagnosis of IVS dissection were found and confirmed by cardiac surgery. The purposes of the study were: to determine the value of transthoracic echocardiography (TTE) in establishing the diagnosis of IVS dissection, and to detail the TTE features of IVS dissection. Thirteen patients with IVS dissection diagnosed by TTE, 8 males and 5 females were taken from 789,114 TTE studies performed between 1985 and 2014. All underwent cardiac surgery during which their diagnosis was confirmed. The etiology, location, 2-dimensional morphology, and color Doppler findings of IVS dissection were noted. The right sinus of Valsalva (SOV) was involved in 11 of the 13 patients. In 5 patients, a single aneurysm of the right SOV was seen dissecting into the IVS. One patient with a combination of a bicuspid aortic valve and a right SOV aneurysm dissected into the IVS. In 4 patients, aortic valve infective endocarditis resulted in IVS dissection. In 1 patient, mechanical aortic valve prosthetic replacement was complicated by annular detachment and a severe paravalvular leak causing IVS dissection. In all 11 patients, TTE showed a dissecting cystic-like mass in the IVS from the base to the mid-septum or confined to the septal base. The path of the dissection in these 11 patients was traced to the right SOV and communications between the IVS dissection and the aortic root were identified. In the remaining 2 patients, IVS dissection followed septal rupture due to a myocardial infarction, and communication was seen between the IVS dissection and the right ventricle. The study showed that most of the dissections of the IVS commence in the right SOV, due to either congenital anomalies or infective endocarditis, or following aortic valve replacement or myocardial infarction. The TTE characteristic of IVS dissection is

  15. Dissecting Diversity Part II

    ERIC Educational Resources Information Center

    Matthews, Frank

    2005-01-01

    This article presents "Dissecting Diversity, Part II," the conclusion of a wide-ranging two-part roundtable discussion on diversity in higher education. The participants were as follows: Lezli Baskerville, J.D., President and CEO of the National Association for Equal Opportunity (NAFEO); Dr. Gerald E. Gipp, Executive Director of the…

  16. Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation.

    PubMed

    Tan, Haiyan; Yang, Kai; Li, Yuxin; Shaw, Timothy I; Wang, Yanyan; Blanco, Daniel Bastardo; Wang, Xusheng; Cho, Ji-Hoon; Wang, Hong; Rankin, Sherri; Guy, Cliff; Peng, Junmin; Chi, Hongbo

    2017-03-21

    The molecular circuits by which antigens activate quiescent T cells remain poorly understood. We combined temporal profiling of the whole proteome and phosphoproteome via multiplexed isobaric labeling proteomics technology, computational pipelines for integrating multi-omics datasets, and functional perturbation to systemically reconstruct regulatory networks underlying T cell activation. T cell receptors activated the T cell proteome and phosphoproteome with discrete kinetics, marked by early dynamics of phosphorylation and delayed ribosome biogenesis and mitochondrial activation. Systems biology analyses identified multiple functional modules, active kinases, transcription factors and connectivity between them, and mitochondrial pathways including mitoribosomes and complex IV. Genetic perturbation revealed physiological roles for mitochondrial enzyme COX10-mediated oxidative phosphorylation in T cell quiescence exit. Our multi-layer proteomics profiling, integrative network analysis, and functional studies define landscapes of the T cell proteome and phosphoproteome and reveal signaling and bioenergetics pathways that mediate lymphocyte exit from quiescence.

  17. Alternatives To Dissection. Second Edition.

    ERIC Educational Resources Information Center

    DeRosa, Bill, Ed.; Winiarskyj, Lesia, Ed.

    This packet attempts to provide educationally sound alternatives to dissection in the classroom, thereby making it possible for teachers to eliminate dissection from the curriculum. This packet can also be used by educators who include dissection in their curricula but consider it important to respect the expression of students' ethical, moral, or…

  18. Recent advances in enrichment and separation strategies for mass spectrometry-based phosphoproteomics

    PubMed Central

    Yang, Chenxi; Zhong, Xuefei; Li, Lingjun

    2016-01-01

    Due to the significance of protein phosphorylation in various biological processes and signaling events, new analytical techniques for enhanced phosphoproteomics have been rapidly introduced in recent years. The combinatorial use of the phospho-specific enrichment techniques and prefractionation methods prior to MS analysis enables comprehensive profiling of the phosphoproteome and facilitates deciphering the critical roles that phosphorylation plays in signaling pathways in various biological systems. This review places special emphasis on the recent five-year (2009–2013) advances for enrichment and separation techniques that have been utilized for phosphopeptides prior to MS analysis. PMID:24687451

  19. Large-Scale Proteomics and Phosphoproteomics of Urinary Exosomes

    PubMed Central

    Gonzales, Patricia A.; Pisitkun, Trairak; Hoffert, Jason D.; Tchapyjnikov, Dmitry; Star, Robert A.; Kleta, Robert; Wang, Nam Sun; Knepper, Mark A.

    2009-01-01

    Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/. PMID:19056867

  20. Repair of type A dissection-benefits of dissection rota

    PubMed Central

    Bashir, Mohamad; Shaw, Matthew; Field, Mark; Kuduvalli, Manoj; Harrington, Deborah; Fok, Mathew

    2016-01-01

    Background Acute type A aortic dissection repair is a surgical emergency associated with high mortality. In 2007, Liverpool Heart & Chest Hospital was the first institution in the United Kingdom to implement a thoracic aortic on-call dissection rota. We set out to investigate whether the dissection rota improved hospital quality outcomes and long-term survival. Methods Data from a prospectively collected database was analysed following case note validation. Two hundred patients underwent acute type A aortic dissection repair between October 1998 and November 2015. To assess the effect of the post-dissection rota on operative and postoperative outcomes, propensity matching of pre- and post-dissection rota patients was used. Results Eighty patients were identified from the pre-dissection rota era and 120 from the post-dissection rota era. Sixty patients from each era were then propensity matched. Comparative analyses showed that patients who underwent acute type A dissection repair in the post-dissection rota period were less likely to suffer in-hospital mortality in both the matched and unmatched groups (30% vs. 13.3%; P=0.004 and 28.3% vs. 11.7%; P=0.055, respectively). A similar improvement was shown in acute renal failure (26.3% vs. 14.2%; P=0.033 and 31.7% vs. 15.0%; P=0.044, respectively). However, cardiopulmonary bypass times and aortic cross clamp times were still significantly longer in the matched post–dissection rota cohort. There was a significant improvement in 5-year survival for the pre- and post-dissection rota in both the matched and unmatched patients (P=0.004 and P=0.034). Conclusions Reorganization of surgical expertise, activity and implementation of a dissection rota within our hospital have resulted in lower in-hospital mortality and better survival outcomes in this group of patients. PMID:27386408

  1. Integration of phosphoproteomic, chemical, and biological strategies for the functional analysis of targeted protein phosphorylation.

    PubMed

    Guo, Mingquan; Huang, Bill X

    2013-02-01

    Reversible phosphorylation, tightly controlled by protein kinases and phosphatases, plays a central role in mediating biological processes, such as protein-protein interactions, subcellular translocation, and activation of cellular enzymes. MS-based phosphoproteomics has now allowed the detection and quantification of tens of thousands of phosphorylation sites from a typical biological sample in a single experiment, which has posed new challenges in functional analysis of each and every phosphorylation site on specific signaling phosphoproteins of interest. In this article, we review recent advances in the functional analysis of targeted phosphorylation carried out by various chemical and biological approaches in combination with the MS-based phosphoproteomics. This review focuses on three types of strategies, including forward functional analysis, defined for the result-driven phosphoproteomics efforts in determining the substrates of a specific protein kinase; reverse functional analysis, defined for tracking the kinase(s) for specific phosphosite(s) derived from the discovery-driven phosphoproteomics efforts; and MS-based analysis on the structure-function relationship of phosphoproteins. It is expected that this review will provide a state-of-the-art overview of functional analysis of site-specific phosphorylation and explore new perspectives and outline future challenges.

  2. Synaptic activity bidirectionally regulates a novel sequence-specific S-Q phosphoproteome in neurons

    PubMed Central

    Siddoway, Benjamin; Hou, Hailong; Yang, Hongtian; Petralia, Ronald; Xia, Houhui

    2013-01-01

    Protein phosphorylation plays a critical role in neuronal transcription, translation, cell viability, and synaptic plasticity. In neurons, phospho-enzymes and specific substrates directly link glutamate release and post-synaptic depolarization to these cellular functions; however, many of these enzymes and their protein substrates remain uncharacterized or unidentified. In this article, we identify a novel, synaptically-driven neuronal phosphoproteome characterized by a specific motif of serine/threonine-glutamine ([S/T]-Q, abbreviated as SQ). These SQ-containing substrates are predominantly localized to dendrites, synapses, the soma; and activation of this SQ phosphoproteome by bicuculline application is induced via calcium influx through L-type calcium channels. On the other hand, acute application of NMDA can inactivate this SQ phosphoproteome. We demonstrate that the SQ motif kinase Ataxia-telangiectasia mutated (ATM) can also localize to dendrites and dendritic spines, in addition to other subcellular compartments, and is activated by bicuculline application. Pharmacology studies indicate that ATM and its sister kinase ATR up-regulate these neuronal SQ substrates. Phosphoproteomics identified over 150 SQ-containing substrates whose phosphorylation is bidirectionally-regulated by synaptic activity. PMID:24117848

  3. Wrangling Phosphoproteomic Data to Elucidate Cancer Signaling Pathways

    PubMed Central

    Grimes, Mark L.; Lee, Wan-Jui; van der Maaten, Laurens; Shannon, Paul

    2013-01-01

    The interpretation of biological data sets is essential for generating hypotheses that guide research, yet modern methods of global analysis challenge our ability to discern meaningful patterns and then convey results in a way that can be easily appreciated. Proteomic data is especially challenging because mass spectrometry detectors often miss peptides in complex samples, resulting in sparsely populated data sets. Using the R programming language and techniques from the field of pattern recognition, we have devised methods to resolve and evaluate clusters of proteins related by their pattern of expression in different samples in proteomic data sets. We examined tyrosine phosphoproteomic data from lung cancer samples. We calculated dissimilarities between the proteins based on Pearson or Spearman correlations and on Euclidean distances, whilst dealing with large amounts of missing data. The dissimilarities were then used as feature vectors in clustering and visualization algorithms. The quality of the clusterings and visualizations were evaluated internally based on the primary data and externally based on gene ontology and protein interaction networks. The results show that t-distributed stochastic neighbor embedding (t-SNE) followed by minimum spanning tree methods groups sparse proteomic data into meaningful clusters more effectively than other methods such as k-means and classical multidimensional scaling. Furthermore, our results show that using a combination of Spearman correlation and Euclidean distance as a dissimilarity representation increases the resolution of clusters. Our analyses show that many clusters contain one or more tyrosine kinases and include known effectors as well as proteins with no known interactions. Visualizing these clusters as networks elucidated previously unknown tyrosine kinase signal transduction pathways that drive cancer. Our approach can be applied to other data types, and can be easily adopted because open source software

  4. Phosphoproteomic Profiling of Selenate-Treated Alzheimer's Disease Model Cells

    PubMed Central

    Wang, Yong; Li, Shuiming; Shen, Liming; Liu, Qiong; Ni, Jiazuan

    2014-01-01

    The reversible phosphorylation of proteins regulates most biological processes, while abnormal phosphorylation is a cause or consequence of many diseases including Alzheimer's disease (AD). One of the hallmarks of AD is the formation of neurofibrillary tangles (NFTs), which is composed of hyperphosphorylated tau proteins. Sodium selenate has been recently found to reduce tau hyperphosphorylation and NFTs formation, and to improve spatial learning and motor performance in AD mice. In the current study, the phosphoproteomics of N2aSW cells treated with selenate were investigated. To avoid missing low-abundance phosphoproteins, both the total proteins of cells and the phosphor-enriched proteins were extracted and subjected to the two-dimensional gel electrophoresis with Pro-Q diamond staining and then LC-MS/MS analysis. A total of 65 proteins were altered in phosphorylation level, of which 39 were up-regulated and 26 were down-regulated. All identified phosphoproteins were bioinformatically annotated according to their physiochemical features, subcellular location, and biological function. Most of these significantly changed phosphoproteins are involved in crucial neural processes such as protesome activity, oxidative stress, cysteine and methionine metabolism, and energy metabolism. Furthermore, decreases were found in homocysteine, phosphor-tau and amyloid β upon selenate treatment. Our results suggest that selenate may intervene in the pathological process of AD by altering the phosphorylation of some key proteins involved in oxidative stress, energy metabolism and protein degradation, thus play important roles in maintaining redox homeostasis, generating ATP, and clearing misfolded proteins and aggregates. The present paper provides some new clues to the mechanism of selenate in AD prevention. PMID:25485856

  5. Cortical granule complements in human oocytes undergoing partial zona dissection.

    PubMed

    Lanzendorf, S E; Kazer, R R; Patton, P E; Wolf, D P

    1992-02-01

    This study was performed to evaluate the effects of mechanical stimulation and sucrose treatment on the oocyte activation process. Fresh and aged human oocytes were exposed to sucrose and zonae were dissected with microneedles before fixation and quantitative analysis of cortical granules by transmission electron microscopy. Examination of the mean number of cortical granules/analyzed segment revealed no significant differences between control oocytes or oocytes treated with sucrose or sucrose treatment followed by zona dissection. A significant decline in the number of cortical granules/segment was observed for oocytes undergoing prolonged culture after dissection (P less than 0.05). Thus, zona dissection and sucrose exposure of freshly aspirated mature human oocytes do not result in classical oocyte activation.

  6. Popliteal lymph node dissection.

    PubMed

    Sholar, Alina; Martin, Robert C G; McMasters, Kelly M

    2005-02-01

    Most sentinel nodes are located in the cervical, axillary, and inguinal nodal basins. Sometimes, however, sentinel nodes exist outside these traditional nodal basins. Popliteal nodal metastasis is relatively uncommon, and popliteal lymph node dissection is infrequently necessary. However, with lymphoscintigraphic identification of popliteal sentinel nodes, surgeons are more frequently called on to address the popliteal nodal basin. Therefore, knowledge of the anatomy and surgical technique for popliteal lymphadenectomy is essential. This case study illustrates the importance of considering the approach to the popliteal lymph node basin for patients with melanoma.

  7. Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen.

    PubMed

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper; Arthur, Jonathan W; Graham, Mark E; Lavin, Martin

    2016-03-01

    Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM

  8. Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer.

    PubMed

    Wu, Xinyan; Zahari, Muhammad Saddiq; Renuse, Santosh; Nirujogi, Raja Sekhar; Kim, Min-Sik; Manda, Srikanth S; Stearns, Vered; Gabrielson, Edward; Sukumar, Saraswati; Pandey, Akhilesh

    2015-11-01

    Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers.

  9. Phosphoproteomic profiling of tumor tissues identifies HSP27 Ser82 phosphorylation as a robust marker of early ischemia

    PubMed Central

    Zahari, Muhammad Saddiq; Wu, Xinyan; Pinto, Sneha M.; Nirujogi, Raja Sekhar; Kim, Min-Sik; Fetics, Barry; Philip, Mathew; Barnes, Sheri R.; Godfrey, Beverly; Gabrielson, Edward; Nevo, Erez; Pandey, Akhilesh

    2015-01-01

    Delays between tissue collection and tissue fixation result in ischemia and ischemia-associated changes in protein phosphorylation levels, which can misguide the examination of signaling pathway status. To identify a biomarker that serves as a reliable indicator of ischemic changes that tumor tissues undergo, we subjected harvested xenograft tumors to room temperature for 0, 2, 10 and 30 minutes before freezing in liquid nitrogen. Multiplex TMT-labeling was conducted to achieve precise quantitation, followed by TiO2 phosphopeptide enrichment and high resolution mass spectrometry profiling. LC-MS/MS analyses revealed phosphorylation level changes of a number of phosphosites in the ischemic samples. The phosphorylation of one of these sites, S82 of the heat shock protein 27 kDa (HSP27), was especially abundant and consistently upregulated in tissues with delays in freezing as short as 2 minutes. In order to eliminate effects of ischemia, we employed a novel cryogenic biopsy device which begins freezing tissues in situ before they are excised. Using this device, we showed that the upregulation of phosphorylation of S82 on HSP27 was abrogated. We thus demonstrate that our cryogenic biopsy device can eliminate ischemia-induced phosphoproteome alterations, and measurements of S82 on HSP27 can be used as a robust marker of ischemia in tissues. PMID:26329039

  10. "Dissection" of a Hair Dryer

    ERIC Educational Resources Information Center

    Eisenstein, Stan; Simpson, Jeff

    2008-01-01

    The electrical design of the common hair dryer is based almost entirely on relatively simple principles learned in introductory physics classes. Just as biology students dissect a frog to see the principles of anatomy in action, physics students can "dissect" a hair dryer to see how principles of electricity are used in a real system. They can…

  11. [Stent Grafting for Aortic Dissection].

    PubMed

    Uchida, Naomichi

    2016-07-01

    The purpose of stent graft for aortic dissection is to terminate antegrade blood flow into the false lumen through primary entry. Early intervention for primary entry makes excellent aortic remodeling and emergent stent grafting for complicated acute type B aortic dissection is supported as a class I. On the other hand stent grafting for chronic aortic dissection is controversial. Early stent grafting is considered with in 6 months after on-set if the diameter of the descending aorta is more than 40 mm. Additional interventions for residual false lumen on the downstream aorta are still required. Stent graft for re-entry, candy-plug technique, and double stenting, other effective re-interventions were reported. Best treatment on the basis of each anatomical and physical characteristics should be selected in each institution. Frozen elephant trunk is alternative procedure for aortic dissection without the need to take account of proximal anatomical limitation and effective for acute type A aortic dissection.

  12. Spontaneous coronary artery dissection.

    PubMed

    Giacoppo, Daniele; Capodanno, Davide; Dangas, George; Tamburino, Corrado

    2014-07-15

    Spontaneous coronary artery dissection (SCAD) is a relatively rare and unexplored type of coronary disease. Although atherosclerosis, hormonal changes during pregnancy and connective tissue disorders might represent a sufficiently convincing explanation for some patients with SCAD, the many remaining cases display only a weak relationship with these causes. While on one side the clinical heterogeneity of SCAD masks a full understanding of their underlying pathophysiologic process, on the other side paucity of data and misleading presentations hamper the quick diagnosis and optimal management of this condition. A definite diagnosis of SCAD can be significantly facilitated by endovascular imaging techniques. In fact, intravascular ultrasound (IVUS) and optical coherence tomography (OCT) overcome the limitations of coronary angiography providing detailed endovascular morphologic information. In contrast, optimal treatment strategies for SCAD still represent a burning controversial question. Herein, we review the published data examining possible causes and investigating the best therapy for SCAD in different clinical scenarios.

  13. Dataset of the Botrytis cinerea phosphoproteome induced by different plant-based elicitors.

    PubMed

    Liñeiro, Eva; Chiva, Cristina; Cantoral, Jesús M; Sabido, Eduard; Fernández-Acero, Francisco Javier

    2016-06-01

    Phosphorylation is one of the main post-translational modification (PTM) involved in signaling network in the ascomycete Botrytis cinerea, one of the most relevant phytopathogenic fungus. The data presented in this article provided a differential mass spectrometry-based analysis of the phosphoproteome of B. cinerea under two different phenotypical conditions induced by the use of two different elicitors: glucose and deproteinized Tomate Cell Walls (TCW). A total 1138 and 733 phosphoproteins were identified for glucose and TCW culture conditions respectively. Raw data are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier (PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD003099). Further interpretation and discussion of these data are provided in our research article entitled "Phosphoproteome analysis of B.cinerea in response to different plant-based elicitors" (Liñeiro et al., 2016) [1].

  14. StageTip-based HAMMOC, an efficient and inexpensive phosphopeptide enrichment method for plant shotgun phosphoproteomics.

    PubMed

    Nakagami, Hirofumi

    2014-01-01

    Phosphopeptide enrichment is the most critical step for successful LC-MS/MS-based shotgun phosphoproteomics. Recent technological improvements have made selective phosphopeptide enrichment from non-fractionated whole cell lysate digests with a single-step procedure possible. Here, a handy protocol is described for phosphopeptide enrichment from plant materials using hydroxy acid-modified metal oxide chromatography (HAMMMOC) with a stop-and-go-extraction tip (StageTip).

  15. Phosphoproteome of the cyanobacterium Synechocystis sp. PCC 6803 and its dynamics during nitrogen starvation

    PubMed Central

    Spät, Philipp; Maček, Boris; Forchhammer, Karl

    2015-01-01

    Cyanobacteria have shaped the earth's biosphere as the first oxygenic photoautotrophs and still play an important role in many ecosystems. The ability to adapt to changing environmental conditions is an essential characteristic in order to ensure survival. To this end, numerous studies have shown that bacteria use protein post-translational modifications such as Ser/Thr/Tyr phosphorylation in cell signaling, adaptation, and regulation. Nevertheless, our knowledge of cyanobacterial phosphoproteomes and their dynamic response to environmental stimuli is relatively limited. In this study, we applied gel-free methods and high accuracy mass spectrometry toward the detection of Ser/Thr/Tyr phosphorylation events in the model cyanobacterium Synechocystis sp. PCC 6803. We could identify over 300 phosphorylation events in cultures grown on nitrate as exclusive nitrogen source. Chemical dimethylation labeling was applied to investigate proteome and phosphoproteome dynamics during nitrogen starvation. Our dataset describes the most comprehensive (phospho)proteome of Synechocystis to date, identifying 2382 proteins and 183 phosphorylation events and quantifying 2111 proteins and 148 phosphorylation events during nitrogen starvation. Global protein phosphorylation levels were increased in response to nitrogen depletion after 24 h. Among the proteins with increased phosphorylation, the PII signaling protein showed the highest fold-change, serving as positive control. Other proteins with increased phosphorylation levels comprised functions in photosynthesis and in carbon and nitrogen metabolism. This study reveals dynamics of Synechocystis phosphoproteome in response to environmental stimuli and suggests an important role of protein Ser/Thr/Tyr phosphorylation in fundamental mechanisms of homeostatic control in cyanobacteria. PMID:25873915

  16. The proteome and phosphoproteome of maize pollen uncovers fertility candidate proteins.

    PubMed

    Chao, Qing; Gao, Zhi-Fang; Wang, Yue-Feng; Li, Zhe; Huang, Xia-He; Wang, Ying-Chun; Mei, Ying-Chang; Zhao, Biligen-Gaowa; Li, Liang; Jiang, Yu-Bo; Wang, Bai-Chen

    2016-06-01

    Maize is unique since it is both monoecious and diclinous (separate male and female flowers on the same plant). We investigated the proteome and phosphoproteome of maize pollen containing modified proteins and here we provide a comprehensive pollen proteome and phosphoproteome which contain 100,990 peptides from 6750 proteins and 5292 phosphorylated sites corresponding to 2257 maize phosphoproteins, respectively. Interestingly, among the total 27 overrepresented phosphosite motifs we identified here, 11 were novel motifs, which suggested different modification mechanisms in plants compared to those of animals. Enrichment analysis of pollen phosphoproteins showed that pathways including DNA synthesis/chromatin structure, regulation of RNA transcription, protein modification, cell organization, signal transduction, cell cycle, vesicle transport, transport of ions and metabolisms, which were involved in pollen development, the following germination and pollen tube growth, were regulated by phosphorylation. In this study, we also found 430 kinases and 105 phosphatases in the maize pollen phosphoproteome, among which calcium dependent protein kinases (CDPKs), leucine rich repeat kinase, SNF1 related protein kinases and MAPK family proteins were heavily enriched and further analyzed. From our research, we also uncovered hundreds of male sterility-associated proteins and phosphoproteins that might influence maize productivity and serve as targets for hybrid maize seed production. At last, a putative complex signaling pathway involving CDPKs, MAPKs, ubiquitin ligases and multiple fertility proteins was constructed. Overall, our data provides new insight for further investigation of protein phosphorylation status in mature maize pollen and construction of maize male sterile mutants in the future.

  17. Application of Phosphoproteomics to Find Targets of Casein Kinase 1 in the Flagellum of Chlamydomonas

    PubMed Central

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2012-01-01

    The green biflagellate alga Chlamydomonas reinhardtii serves as model for studying structural and functional features of flagella. The axoneme of C. reinhardtii anchors a network of kinases and phosphatases that control motility. One of them, Casein Kinase 1 (CK1), is known to phosphorylate the Inner Dynein Arm I1 Intermediate Chain 138 (IC138), thereby regulating motility. CK1 is also involved in regulating the circadian rhythm of phototaxis and is relevant for the formation of flagella. By a comparative phosphoproteome approach, we determined phosphoproteins in the flagellum that are targets of CK1. Thereby, we applied the specific CK1 inhibitor CKI-7 that causes significant changes in the flagellum phosphoproteome and reduces the swimming velocity of the cells. In the CKI-7-treated cells, 14 phosphoproteins were missing compared to the phosphoproteome of untreated cells, including IC138, and four additional phosphoproteins had a reduced number of phosphorylation sites. Notably, inhibition of CK1 causes also novel phosphorylation events, indicating that it is part of a kinase network. Among them, Glycogen Synthase Kinase 3 is of special interest, because it is involved in the phosphorylation of key clock components in flies and mammals and in parallel plays an important role in the regulation of assembly in the flagellum. PMID:23316220

  18. Application of phosphoproteomics to find targets of casein kinase 1 in the flagellum of chlamydomonas.

    PubMed

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2012-01-01

    The green biflagellate alga Chlamydomonas reinhardtii serves as model for studying structural and functional features of flagella. The axoneme of C. reinhardtii anchors a network of kinases and phosphatases that control motility. One of them, Casein Kinase 1 (CK1), is known to phosphorylate the Inner Dynein Arm I1 Intermediate Chain 138 (IC138), thereby regulating motility. CK1 is also involved in regulating the circadian rhythm of phototaxis and is relevant for the formation of flagella. By a comparative phosphoproteome approach, we determined phosphoproteins in the flagellum that are targets of CK1. Thereby, we applied the specific CK1 inhibitor CKI-7 that causes significant changes in the flagellum phosphoproteome and reduces the swimming velocity of the cells. In the CKI-7-treated cells, 14 phosphoproteins were missing compared to the phosphoproteome of untreated cells, including IC138, and four additional phosphoproteins had a reduced number of phosphorylation sites. Notably, inhibition of CK1 causes also novel phosphorylation events, indicating that it is part of a kinase network. Among them, Glycogen Synthase Kinase 3 is of special interest, because it is involved in the phosphorylation of key clock components in flies and mammals and in parallel plays an important role in the regulation of assembly in the flagellum.

  19. Comparative Phosphoproteomics Reveals an Important Role of MKK2 in Banana (Musa spp.) Cold Signal Network

    PubMed Central

    Gao, Jie; Zhang, Sheng; He, Wei-Di; Shao, Xiu-Hong; Li, Chun-Yu; Wei, Yue-Rong; Deng, Gui-Ming; Kuang, Rui-Bin; Hu, Chun-Hua; Yi, Gan-Jun; Yang, Qiao-Song

    2017-01-01

    Low temperature is one of the key environmental stresses, which greatly affects global banana production. However, little is known about the global phosphoproteomes in Musa spp. and their regulatory roles in response to cold stress. In this study, we conducted a comparative phosphoproteomic profiling of cold-sensitive Cavendish Banana and relatively cold tolerant Dajiao under cold stress. Phosphopeptide abundances of five phosphoproteins involved in MKK2 interaction network, including MKK2, HY5, CaSR, STN7 and kinesin-like protein, show a remarkable difference between Cavendish Banana and Dajiao in response to cold stress. Western blotting of MKK2 protein and its T31 phosphorylated peptide verified the phosphoproteomic results of increased T31 phosphopeptide abundance with decreased MKK2 abundance in Daojiao for a time course of cold stress. Meanwhile increased expression of MKK2 with no detectable T31 phosphorylation was found in Cavendish Banana. These results suggest that the MKK2 pathway in Dajiao, along with other cold-specific phosphoproteins, appears to be associated with the molecular mechanisms of high tolerance to cold stress in Dajiao. The results also provide new evidence that the signaling pathway of cellular MKK2 phosphorylation plays an important role in abiotic stress tolerance that likely serves as a universal plant cold tolerance mechanism. PMID:28106078

  20. Comparative Phosphoproteomics Reveals an Important Role of MKK2 in Banana (Musa spp.) Cold Signal Network.

    PubMed

    Gao, Jie; Zhang, Sheng; He, Wei-Di; Shao, Xiu-Hong; Li, Chun-Yu; Wei, Yue-Rong; Deng, Gui-Ming; Kuang, Rui-Bin; Hu, Chun-Hua; Yi, Gan-Jun; Yang, Qiao-Song

    2017-01-20

    Low temperature is one of the key environmental stresses, which greatly affects global banana production. However, little is known about the global phosphoproteomes in Musa spp. and their regulatory roles in response to cold stress. In this study, we conducted a comparative phosphoproteomic profiling of cold-sensitive Cavendish Banana and relatively cold tolerant Dajiao under cold stress. Phosphopeptide abundances of five phosphoproteins involved in MKK2 interaction network, including MKK2, HY5, CaSR, STN7 and kinesin-like protein, show a remarkable difference between Cavendish Banana and Dajiao in response to cold stress. Western blotting of MKK2 protein and its T31 phosphorylated peptide verified the phosphoproteomic results of increased T31 phosphopeptide abundance with decreased MKK2 abundance in Daojiao for a time course of cold stress. Meanwhile increased expression of MKK2 with no detectable T31 phosphorylation was found in Cavendish Banana. These results suggest that the MKK2 pathway in Dajiao, along with other cold-specific phosphoproteins, appears to be associated with the molecular mechanisms of high tolerance to cold stress in Dajiao. The results also provide new evidence that the signaling pathway of cellular MKK2 phosphorylation plays an important role in abiotic stress tolerance that likely serves as a universal plant cold tolerance mechanism.

  1. Phosphoproteomic analysis of protein phosphorylation networks in Tetrahymena thermophila, a model single-celled organism.

    PubMed

    Tian, Miao; Chen, Xiulan; Xiong, Qian; Xiong, Jie; Xiao, Chuanle; Ge, Feng; Yang, Fuquan; Miao, Wei

    2014-02-01

    Tetrahymena thermophila is a widely used unicellular eukaryotic model organism in biological research and contains more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues. However, only a few dozen phosphorylation sites in T. thermophila are known, presenting a major obstacle to further understanding of the regulatory roles of reversible phosphorylation in this organism. In this study, we used high-accuracy mass-spectrometry-based proteomics to conduct global and site-specific phosphoproteome profiling of T. thermophila. In total, 1384 phosphopeptides and 2238 phosphorylation sites from 1008 T. thermophila proteins were identified through the combined use of peptide prefractionation, TiO2 enrichment, and two-dimensional LC-MS/MS analysis. The identified phosphoproteins are implicated in the regulation of various biological processes such as transport, gene expression, and mRNA metabolic process. Moreover, integrated analysis of the T. thermophila phosphoproteome and gene network revealed the potential biological functions of many previously unannotated proteins and predicted some putative kinase-substrate pairs. Our data provide the first global survey of phosphorylation in T. thermophila using a phosphoproteomic approach and suggest a wide-ranging regulatory scope of this modification. The provided dataset is a valuable resource for the future understanding of signaling pathways in this important model organism.

  2. Phosphoproteome Analysis Links Protein Phosphorylation to Cellular Remodeling and Metabolic Adaptation during Magnaporthe oryzae Appressorium Development.

    PubMed

    Franck, William L; Gokce, Emine; Randall, Shan M; Oh, Yeonyee; Eyre, Alex; Muddiman, David C; Dean, Ralph A

    2015-06-05

    The rice pathogen, Magnaporthe oryzae, undergoes a complex developmental process leading to formation of an appressorium prior to plant infection. In an effort to better understand phosphoregulation during appressorium development, a mass spectrometry based phosphoproteomics study was undertaken. A total of 2924 class I phosphosites were identified from 1514 phosphoproteins from mycelia, conidia, germlings, and appressoria of the wild type and a protein kinase A (PKA) mutant. Phosphoregulation during appressorium development was observed for 448 phosphosites on 320 phosphoproteins. In addition, a set of candidate PKA targets was identified encompassing 253 phosphosites on 227 phosphoproteins. Network analysis incorporating regulation from transcriptomic, proteomic, and phosphoproteomic data revealed new insights into the regulation of the metabolism of conidial storage reserves and phospholipids, autophagy, actin dynamics, and cell wall metabolism during appressorium formation. In particular, protein phosphorylation appears to play a central role in the regulation of autophagic recycling and actin dynamics during appressorium formation. Changes in phosphorylation were observed in multiple components of the cell wall integrity pathway providing evidence that this pathway is highly active during appressorium development. Several transcription factors were phosphoregulated during appressorium formation including the bHLH domain transcription factor MGG_05709. Functional analysis of MGG_05709 provided further evidence for the role of protein phosphorylation in regulation of glycerol metabolism and the metabolic reprogramming characteristic of appressorium formation. The data presented here represent a comprehensive investigation of the M. oryzae phosphoproteome and provide key insights on the role of protein phosphorylation during infection-related development.

  3. Phosphoproteomic Analysis of Platelets Activated by Pro-Thrombotic Oxidized Phospholipids and Thrombin

    PubMed Central

    Zimman, Alejandro; Titz, Bjoern; Komisopoulou, Evangelia; Biswas, Sudipta; Graeber, Thomas G.; Podrez, Eugene A.

    2014-01-01

    Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36. PMID:24400094

  4. Phosphoproteomic Analysis of Protein Kinase C Signaling in Saccharomyces cerevisiae Reveals Slt2 Mitogen-activated Protein Kinase (MAPK)-dependent Phosphorylation of Eisosome Core Components*

    PubMed Central

    Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J.; Molina, María

    2013-01-01

    The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1–cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module. PMID:23221999

  5. Identification of the PLK2-Dependent Phosphopeptidome by Quantitative Proteomics

    PubMed Central

    Franchin, Cinzia; Cesaro, Luca; Pinna, Lorenzo A.; Arrigoni, Giorgio; Salvi, Mauro

    2014-01-01

    Polo-like kinase 2 (PLK2) has been recently recognized as the major enzyme responsible for phosphorylation of α-synuclein at S129 in vitro and in vivo, suggesting that this kinase may play a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Moreover PLK2 seems to be implicated in cell division, oncogenesis, and synaptic regulation of the brain. However little is known about the phosphoproteome generated by PLK2 and, consequently the overall impact of PLK2 on cellular signaling. To fill this gap we exploited an approach based on in vitro kinase assay and quantitative phosphoproteomics. A proteome-derived peptide library obtained by digestion of undifferentiated human neuroblastoma cell line was exhaustively dephosphorylated by lambda phosphatase followed by incubation with or without PLK2 recombinant kinase. Stable isotope labeling based quantitative phosphoproteomics was applied to identify the phosphosites generated by PLK2. A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified by LC-MS/MS. Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated database of in cell/vivo phosphorylation sites. PMID:25338102

  6. Triple quad ICPMS (ICPQQQ) as a new tool for absolute quantitative proteomics and phosphoproteomics.

    PubMed

    Diez Fernández, Silvia; Sugishama, Naoki; Ruiz Encinar, Jorge; Sanz-Medel, Alfredo

    2012-07-17

    It is clear that sensitive and interference-free quantification of ICP-detectable elements naturally present in proteins will boost the role of ICPMS in proteomics. In this study, a completely new way of polyatomic interference removal in ICPMS for detection of sulfur (present in the majority of proteins as methionine or cysteine) and phosphorus (present in phosphorylated proteins) is presented. It is based on the concept of tandem mass spectrometry (QQQ) typically used in molecular MS. Briefly, the first quadrupole can be operated as 1 amu window band-pass mass filter to select target analyte ions ((31)P, (32)S, and their on-mass polyatomic interferences). In this way, only selected ions enter the cell and react with O(2), reducing the interferences produced by matrix ions as well as background noise. After optimization of the cell conditions, product ions formed for the targets, (47)PO(+) and (48)SO(+), could be detected with enhanced sensitivity and selectivity. The coupling to capillary HPLC allowed analysis of S- and P-containing species with the lowest detection limits ever published (11 and 6.6 fmol, respectively). The potential of the approach for proteomics studies was demonstrated for the highly sensitive simultaneous absolute quantification of different S-containing peptides and phosphopeptides.

  7. Spontaneous Arterial Dissection.

    PubMed

    Brandt, Tobias; Caplan, Louis

    2001-09-01

    There is no controlled study for the best treatment or management of cervico-cerebral artery dissection (CAD). Rationale initial empiric treatment in acute CAD to prevent secondary embolism is partial thromboplastin time (PTT)-guided anticoagulation by intravenous heparin followed by anticoagulation with warfarin. Carotid surgery for treatment of CAD is not recommended anymore with the possible exception of persisting severe stenosis of the proximal internal carotid artery (ICA). There could be use of carotid angioplasty by balloon dilatation and stenting in selected cases of severe cerebral hemodynamic impairment by bilateral CAD. Duration of secondary prophylaxis by anticoagulation is best guided by Doppler sonography follow-up, and should be continued until normalization of blood flow or until at least 1 year after the vessel is occluded. There is no evidence that pseudoaneurysms increase the risk for embolic complication, and there is no evidence for surgery or continuation of anticoagulation in patients with pseudoaneurysms. Caution should be recommended for exercises that involve excessive head movements (eg, bungee jumping, trampoline jumping, and chiropractic maneuvers). The patient should be informed that recurrent rate is low in nonfamilial cases. Doppler sonography is a low-cost and high-sensitivity method for patients at risk.

  8. Biomedicine: an ontological dissection.

    PubMed

    Baronov, David

    2008-01-01

    Though ubiquitous across the medical social sciences literature, the term "biomedicine" as an analytical concept remains remarkably slippery. It is argued here that this imprecision is due in part to the fact that biomedicine is comprised of three interrelated ontological spheres, each of which frames biomedicine as a distinct subject of investigation. This suggests that, depending upon one's ontological commitment, the meaning of biomedicine will shift. From an empirical perspective, biomedicine takes on the appearance of a scientific enterprise and is defined as a derivative category of Western science more generally. From an interpretive perspective, biomedicine represents a symbolic-cultural expression whose adherence to the principles of scientific objectivity conceals an ideological agenda. From a conceptual perspective, biomedicine represents an expression of social power that reflects structures of power and privilege within capitalist society. No one perspective exists in isolation and so the image of biomedicine from any one presents an incomplete understanding. It is the mutually-conditioning interrelations between these ontological spheres that account for biomedicine's ongoing development. Thus, the ontological dissection of biomedicine that follows, with particular emphasis on the period of its formal crystallization in the latter nineteenth and early twentieth century, is intended to deepen our understanding of biomedicine as an analytical concept across the medical social sciences literature.

  9. Experience with parametric binary dissection

    NASA Technical Reports Server (NTRS)

    Bokhari, Shahid H.

    1993-01-01

    Parametric Binary Dissection (PBD) is a new algorithm that can be used for partitioning graphs embedded in 2- or 3-dimensional space. It partitions explicitly on the basis of nodes + (lambda)x(edges cut), where lambda is the ratio of time to communicate over an edge to the time to compute at a node. The new algorithm is faster than the original binary dissection algorithm and attempts to obtain better partitions than the older algorithm, which only takes nodes into account. The performance of parametric dissection with plain binary dissection on 3 large unstructured 3-d meshes obtained from computational fluid dynamics and on 2 random graphs were compared. It was showm that the new algorithm can usually yield partitions that are substantially superior, but that its performance is heavily dependent on the input data.

  10. Early history of neck dissection.

    PubMed

    Rinaldo, Alessandra; Ferlito, Alfio; Silver, Carl E

    2008-12-01

    With the exception of distant metastases, the presence of lymph node metastasis in the neck is accepted as the single most important adverse independent prognostic factor and an indicator of survival in squamous carcinoma of the head and neck. Neck dissection in its various forms is the standard surgical treatment for clinical, subclinical and subpathologic metastatic cancer to the neck. The pertinent literature from the beginning of the nineteenth century to the middle of the twentieth century was reviewed. The four giants of late nineteenth century surgery: von Langenbeck, Billroth, von Volkmann and Kocher developed and reported the early cases of different types of neck dissection. Butlin, in England, conceived and developed the concept of elective neck dissection. In 1888, the Polish surgeon Jawdyńsky reported and described in detail the first successful extended en bloc neck dissection. Crile, in 1905 and 1906, reported the first significant series of radical en bloc neck dissections, bringing this procedure to the attention of the medical world as an effective operation with reproducible technique and results. The greatest impetus to the status of this surgical procedure came from Martin and colleagues, who published a monumental report in 1951 of 1,450 cases that established the place and technique of radical neck dissection in the modern treatment of head and neck cancer. Neck dissection, for treatment of cervical lymph node metastases in head and neck cancer, was conceived and attempted in the nineteenth century, with some limited success reported by the end of that era. An effective operation was described and reported in the early twentieth century and evolved by the mid century into a fundamental tool in the management of patients with head and neck cancer.

  11. Triple-barrel aorta: dissection of a healed aortic dissection.

    PubMed

    Lie, J T

    1982-08-01

    An unusual case of a triple-barrel aorta in a 51-year-old woman is described. The patient first had a spontaneous type I dissection of the aorta and acute aortic insufficiency, for which she underwent aortic valve replacement and Dacron graft replacement of the ascending aorta. She remained asymptomatic for five years with a healed aortic dissection (double-barrel aorta) distal to the graft. She then underwent a second operation for repair and poppet replacement of the malfunctioned prosthesis. Postoperative cardiac failure necessitated the use of a counterpulsation intra-aortic balloon catheter, which entered and dissected the wall of the false lumen, thus creating a triple-lumen aorta.

  12. Nuclear phosphoproteome analysis of 3T3-L1 preadipocyte differentiation reveals system-wide phosphorylation of transcriptional regulators.

    PubMed

    Rabiee, Atefeh; Schwämmle, Veit; Sidoli, Simone; Dai, Jie; Rogowska-Wrzesinska, Adelina; Mandrup, Susanne; Jensen, Ole N

    2017-03-01

    Adipocytes (fat cells) are important endocrine and metabolic cells critical for systemic insulin sensitivity. Both adipose excess and insufficiency are associated with adverse metabolic function. Adipogenesis is the process whereby preadipocyte precursor cells differentiate into lipid-laden mature adipocytes. This process is driven by a network of transcriptional regulators (TRs). We hypothesized that protein PTMs, in particular phosphorylation, play a major role in activating and propagating signals within TR networks upon induction of adipogenesis by extracellular stimulus. We applied MS-based quantitative proteomics and phosphoproteomics to monitor the alteration of nuclear proteins during the early stages (4 h) of preadipocyte differentiation. We identified a total of 4072 proteins including 2434 phosphorylated proteins, a majority of which were assigned as regulators of gene expression. Our results demonstrate that adipogenic stimuli increase the nuclear abundance and/or the phosphorylation levels of proteins involved in gene expression, cell organization, and oxidation-reduction pathways. Furthermore, proteins acting as negative modulators involved in negative regulation of gene expression, insulin stimulated glucose uptake, and cytoskeletal organization showed a decrease in their nuclear abundance and/or phosphorylation levels during the first 4 h of adipogenesis. Among 288 identified TRs, 49 were regulated within 4 h of adipogenic stimulation including several known and many novel potential adipogenic regulators. We created a kinase-substrate database for 3T3-L1 preadipocytes by investigating the relationship between protein kinases and protein phosphorylation sites identified in our dataset. A majority of the putative protein kinases belong to the cyclin-dependent kinase family and the mitogen-activated protein kinase family including P38 and c-Jun N-terminal kinases, suggesting that these kinases act as orchestrators of early adipogenesis.

  13. Combined Quantification of the Global Proteome, Phosphoproteome, and Proteolytic Cleavage to Characterize Altered Platelet Functions in the Human Scott Syndrome*

    PubMed Central

    Solari, Fiorella A.; Mattheij, Nadine J.A.; Burkhart, Julia M.; Swieringa, Frauke; Collins, Peter W.; Cosemans, Judith M.E.M.; Sickmann, Albert; Heemskerk, Johan W.M.; Zahedi, René P.

    2016-01-01

    The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca2+-dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca2+-dependent changes that are normally associated with phosphatidylserine exposure. PMID:27535140

  14. Online, absolute quantitation of propranolol from spatially distinct 20-μm and 40-μm dissections of brain, liver, and kidney thin tissue sections by laser microdissection – liquid vortex capture – mass spectrometry

    DOE PAGES

    Kertesz, Vilmos; Vavrek, Marissa; Freddo, Carol; ...

    2016-05-23

    Here, spatial resolved quantitation of chemical species in thin tissue sections by mass spectrometric methods has been constrained by the need for matrix-matched standards or other arduous calibration protocols and procedures to mitigate matrix effects (e.g., spatially varying ionization suppression). Reported here is the use of laser cut and drop sampling with a laser microdissection-liquid vortex capture electrospray ionization tandem mass spectrometry (LMD-LVC/ESI-MS/MS) system for online and absolute quantitation of propranolol in mouse brain, kidney, and liver thin tissue sections of mice administered with the drug at a 7.5 mg/kg dose, intravenously. In this procedure either 20 μm x 20more » μm or 40 μm x 40 μm tissue microdissections were cut and dropped into the flowing solvent of the capture probe. During transport to the ESI source drug related material was completely extracted from the tissue into the solvent, which contained a known concentration of propranolol-d7 as an internal standard. This allowed absolute quantitation to be achieved with an external calibration curve generated from standards containing the same fixed concentration of propranolold-d7 and varied concentrations of propranolol. Average propranolol concentrations determined with the laser cut and drop sampling method closely agreed with concentration values obtained from 2.3 mm diameter tissue punches from serial sections that were extracted and quantified by HPLC/ESI-MS/MS measurements. In addition, the relative abundance of hydroxypropranolol glucuronide metabolites were recorded and found to be consistent with previous findings.« less

  15. Online, absolute quantitation of propranolol from spatially distinct 20-μm and 40-μm dissections of brain, liver, and kidney thin tissue sections by laser microdissection – liquid vortex capture – mass spectrometry

    SciTech Connect

    Kertesz, Vilmos; Vavrek, Marissa; Freddo, Carol; Van Berkel, Gary J.; Cahill, John F.; Weiskittel, Taylor M.

    2016-05-23

    Here, spatial resolved quantitation of chemical species in thin tissue sections by mass spectrometric methods has been constrained by the need for matrix-matched standards or other arduous calibration protocols and procedures to mitigate matrix effects (e.g., spatially varying ionization suppression). Reported here is the use of laser cut and drop sampling with a laser microdissection-liquid vortex capture electrospray ionization tandem mass spectrometry (LMD-LVC/ESI-MS/MS) system for online and absolute quantitation of propranolol in mouse brain, kidney, and liver thin tissue sections of mice administered with the drug at a 7.5 mg/kg dose, intravenously. In this procedure either 20 μm x 20 μm or 40 μm x 40 μm tissue microdissections were cut and dropped into the flowing solvent of the capture probe. During transport to the ESI source drug related material was completely extracted from the tissue into the solvent, which contained a known concentration of propranolol-d7 as an internal standard. This allowed absolute quantitation to be achieved with an external calibration curve generated from standards containing the same fixed concentration of propranolold-d7 and varied concentrations of propranolol. Average propranolol concentrations determined with the laser cut and drop sampling method closely agreed with concentration values obtained from 2.3 mm diameter tissue punches from serial sections that were extracted and quantified by HPLC/ESI-MS/MS measurements. In addition, the relative abundance of hydroxypropranolol glucuronide metabolites were recorded and found to be consistent with previous findings.

  16. Phosphoproteomic analysis of the Chlamydia caviae elementary body and reticulate body forms.

    PubMed

    Fisher, Derek J; Adams, Nancy E; Maurelli, Anthony T

    2015-08-01

    Chlamydia are Gram-negative, obligate intracellular bacteria responsible for significant diseases in humans and economically important domestic animals. These pathogens undergo a unique biphasic developmental cycle transitioning between the environmentally stable elementary body (EB) and the replicative intracellular reticulate body (RB), a conversion that appears to require extensive regulation of protein synthesis and function. However, Chlamydia possess a limited number of canonical mechanisms of transcriptional regulation. Ser/Thr/Tyr phosphorylation of proteins in bacteria has been increasingly recognized as an important mechanism of post-translational control of protein function. We utilized 2D gel electrophoresis coupled with phosphoprotein staining and MALDI-TOF/TOF analysis to map the phosphoproteome of the EB and RB forms of Chlamydia caviae. Forty-two non-redundant phosphorylated proteins were identified (some proteins were present in multiple locations within the gels). Thirty-four phosphorylated proteins were identified in EBs, including proteins found in central metabolism and protein synthesis, Chlamydia-specific hypothetical proteins and virulence-related proteins. Eleven phosphorylated proteins were identified in RBs, mostly involved in protein synthesis and folding and a single virulence-related protein. Only three phosphoproteins were found in both EB and RB phosphoproteomes. Collectively, 41 of 42 C. caviae phosphoproteins were present across Chlamydia species, consistent with the existence of a conserved chlamydial phosphoproteome. The abundance of stage-specific phosphoproteins suggests that protein phosphorylation may play a role in regulating the function of developmental-stage-specific proteins and/or may function in concert with other factors in directing EB-RB transitions.

  17. [Pregnancy and coronary artery dissection].

    PubMed

    Martínez-Quintana, Efrén; Rodríguez-González, Fayna

    2015-01-01

    Acute myocardial infarction during pregnancy is associated with high maternal and fetal mortality. Coronary atherosclerosis is the most common cause due to an increase in the age of the patients and the association with cardiovascular risk factors such as smoking, hypertension, diabetes mellitus, preeclampsia, and the existence of family history of coronary disease. However, thrombosis, coronary dissection or coronary vasospasms are other causes that may justify it. We report the case of a 33 weeks pregnant first-time mother, without cardiovascular risk factors, who presented an acute coronary event in the context of atherosclerotic disease and coronary dissection after percutaneous coronary intervention.

  18. ``Dissection'' of a Hair Dryer

    NASA Astrophysics Data System (ADS)

    Eisenstein, Stan; Simpson, Jeff

    2008-12-01

    The electrical design of the common hair dryer is based almost entirely on relatively simple principles learned in introductory physics classes. Just as biology students dissect a frog to see the principles of anatomy in action, physics students can "dissect" a hair dryer to see how principles of electricity are used in a real system. They can discover how engineers solve problems such as how to vary between low and high heat and fan speed by simply moving the position of a single switch. Principles of alternating versus direct current, series and parallel circuits, electrical safety, voltage dividing, ac rectification, power, and measurement of resistance and continuity all come in to play.

  19. Diagnostic imaging for aortic dissection.

    PubMed

    Kapustin, Andrew J; Litt, Harold I

    2005-01-01

    Diagnostic imaging for aortic dissection has dramatically changed in recent years. Previously, imaging consisted of conventional X-ray radiography, followed by invasive catheter angiography. Now imaging of dissection is performed primarily with multidetector CT, and to a lesser extent, with ultrasound and MRI. Catheter angiography is used primarily as a means of treating complications. Which modality to choose depends on patient factors, physician preference, and differences in availability of state-of-the-art equipment. All three modalities are highly accurate in experienced hands and have revolutionized the detection and evaluation of this condition.

  20. Dissect Your Squid and Eat It Too!

    ERIC Educational Resources Information Center

    McGinnis, Patricia

    2001-01-01

    Introduces a science lab activity in which students dissect fresh squids in groups of four and observe the anatomy. Parent volunteers cook the squid mantle for kids to taste. Includes directions for squid dissection. (YDS)

  1. [Dissection is still important when learning anatomy].

    PubMed

    Knudsen, Britt Mejer; Søe, Niels H; Jensen, Nina Vendel; Langebæk, Rikke; Dahlin, Lars B

    2013-05-20

    Dissection and prosection require a donation programme of cadavers for education and research. The importance of maintaining the donation programme and the significance of dissection as a teaching method when learning anatomic structures and obtaining surgical skills are evaluated.

  2. Dissection & Science Fairs. [Information Packet.

    ERIC Educational Resources Information Center

    National Anti-Vivisection Society, Chicago, IL.

    This collection of pamphlets and articles reprinted from other National Anti-Vivisection Society (NAVS) publications was compiled to address the issues of classroom laboratory dissection and the use of animals in science fair projects. Three of the pamphlets contained in this packet are student handbooks designed to help students of elementary,…

  3. Animal Rights Activism Threatens Dissection.

    ERIC Educational Resources Information Center

    Holden, Constance

    1990-01-01

    Discussed is the movement against the use of dissections in science laboratories. Examples of protests across the United States are included. Compared is the plight of using animals in a biology classroom and the demise of the teaching of evolution in some areas. (KR)

  4. LoQAtE—Localization and Quantitation ATlas of the yeast proteomE. A new tool for multiparametric dissection of single-protein behavior in response to biological perturbations in yeast

    PubMed Central

    Breker, Michal; Gymrek, Melissa; Moldavski, Ofer; Schuldiner, Maya

    2014-01-01

    Living organisms change their proteome dramatically to sustain a stable internal milieu in fluctuating environments. To study the dynamics of proteins during stress, we measured the localization and abundance of the Saccharomyces cerevisiae proteome under various growth conditions and genetic backgrounds using the GFP collection. We created a database (DB) called ‘LoQAtE’ (Localizaiton and Quantitation Atlas of the yeast proteomE), available online at http://www.weizmann.ac.il/molgen/loqate/, to provide easy access to these data. Using LoQAtE DB, users can get a profile of changes for proteins of interest as well as querying advanced intersections by either abundance changes, primary localization or localization shifts over the tested conditions. Currently, the DB hosts information on 5330 yeast proteins under three external perturbations (DTT, H2O2 and nitrogen starvation) and two genetic mutations [in the chaperonin containing TCP1 (CCT) complex and in the proteasome]. Additional conditions will be uploaded regularly. The data demonstrate hundreds of localization and abundance changes, many of which were not detected at the level of mRNA. LoQAtE is designed to allow easy navigation for non-experts in high-content microscopy and data are available for download. These data should open up new perspectives on the significant role of proteins while combating external and internal fluctuations. PMID:24150937

  5. Dissection of the major hematopoietic quantitative trait locus in chromosome 6q23.3 identifies miR-3662 as a novel player in hematopoiesis and acute myeloid leukemia

    PubMed Central

    Maharry, Sophia E.; Walker, Christopher J.; Liyanarachchi, Sandya; Mehta, Sujay; Patel, Mitra; Bainazar, Maryam A.; Huang, Xiaomeng; Lankenau, Malori A.; Hoag, Kevin W.; Ranganathan, Parvathi; Garzon, Ramiro; Blachly, James S.; Guttridge, Denis C.; Bloomfield, Clara D.; de la Chapelle, Albert; Eisfeld, Ann-Kathrin

    2016-01-01

    Chromosomal aberrations and multiple genome-wide association studies (GWASs) have established a major hematopoietic quantitative trait locus in chromosome 6q23.3. The locus comprises an active enhancer region, in which some of the associated SNPs alter transcription factor binding. We now identify microRNA-3662 as a new functional driver contributing to the associated phenotypes. The GWAS SNPs are strongly associated with higher miR-3662 expression. Genome editing of rs66650371, a three base pair deletion, suggests a functional link between the SNP genotype and the abundance of miR-3662. Increasing miR-3662’s abundance increases colony formation in hematopoietic progenitor cells, particularly the erythroid lineage. In contrast, miR-3662 is not expressed in acute myeloid leukemia cells and its overexpression has potent anti-leukemic effects in vitro and in vivo. Mechanistically, miR-3662 directly targets NF-ĸB-mediated transcription. Thus, miR-3662 is a new player of the hematopoietic 6q23.3 locus. PMID:27354268

  6. Concerted Changes in the Phosphoproteome and Metabolome Under Different CO2/O2 Gaseous Conditions in Arabidopsis Rosettes.

    PubMed

    Abadie, Cyril; Mainguet, Samuel; Davanture, Marlène; Hodges, Michael; Zivy, Michel; Tcherkez, Guillaume

    2016-07-01

    Considerable efforts are currently devoted to understanding the regulation of primary carbon metabolism in plant leaves, which is known to change dramatically with environmental conditions, e.g. during light/dark transitions. Protein phosphorylation is believed to be a key factor in such a metabolic control. In fact, some studies have suggested modifications in the phosphorylation status of key enzymes in the dark compared with the light, or when photosynthesis varies. However, a general view of the phosphoproteome and reciprocal alterations in both the phosphoproteome and metabolome under a wide spectrum of CO2 and O2 conditions so as to vary both gross photosynthesis and photorespiration is currently lacking. Here, we used an instant sampling system and strictly controlled gaseous conditions to examine short-term metabolome and phosphoproteome changes in Arabidopsis rosettes. We show that light/dark, CO2 and O2 mole fraction have differential effects on enzyme phosphorylation. Phosphorylation events that appear to be the most important to regulate metabolite contents when photosynthesis varies are those associated with sugar and pyruvate metabolism: sucrose and starch synthesis are major phosphorylation-controlled steps but pyruvate utilization (by phosphoenolpyruvate carboxylase and pyruvate dehydrogenase) and pyruvate reformation (by pyruvate orthophosphate dikinase) are also subjected to phosphorylation control. Our results thus show that the phosphoproteome response to light/dark transition and gaseous conditions (CO2, O2) contributes to the rapid adjustment of major pathways of primary C metabolism.

  7. SELPHI: correlation-based identification of kinase-associated networks from global phospho-proteomics data sets

    PubMed Central

    Petsalaki, Evangelia; Helbig, Andreas O.; Gopal, Anjali; Pasculescu, Adrian; Roth, Frederick P.; Pawson, Tony

    2015-01-01

    While phospho-proteomics studies have shed light on the dynamics of cellular signaling, they mainly describe global effects and rarely explore mechanistic details, such as kinase/substrate relationships. Tools and databases, such as NetworKIN and PhosphoSitePlus, provide valuable regulatory details on signaling networks but rely on prior knowledge. They therefore provide limited information on less studied kinases and fewer unexpected relationships given that better studied signaling events can mask condition- or cell-specific ‘network wiring’. SELPHI is a web-based tool providing in-depth analysis of phospho-proteomics data that is intuitive and accessible to non-bioinformatics experts. It uses correlation analysis of phospho-sites to extract kinase/phosphatase and phospho-peptide associations, and highlights the potential flow of signaling in the system under study. We illustrate SELPHI via analysis of phospho-proteomics data acquired in the presence of erlotinib—a tyrosine kinase inhibitor (TKI)—in cancer cells expressing TKI-resistant and -sensitive variants of the Epidermal Growth Factor Receptor. In this data set, SELPHI revealed information overlooked by the reporting study, including the known role of MET and EPHA2 kinases in conferring resistance to erlotinib in TKI sensitive strains. SELPHI can significantly enhance the analysis of phospho-proteomics data contributing to improved understanding of sample-specific signaling networks. SELPHI is freely available via http://llama.mshri.on.ca/SELPHI. PMID:25948583

  8. Cutting Edge Controversy: The Politics of Animal Dissection and Responses to Student Objection

    NASA Astrophysics Data System (ADS)

    Oakley, Jan

    2011-12-01

    This mixed methods study investigated the experiences and perspectives of former Ontario high school students and current Ontario science and biology teachers toward animal dissection, objection to dissection, and choice policies that grant students the right to opt out of dissection and use an alternative instead. Data was collected via a student questionnaire (n=311), a teacher questionnaire (n=153), and interviews with eight students and nine teachers. Quantitative and qualitative data analyses and reporting techniques were employed within a humane education and critical pedagogy framework to explore the experiences and perspectives of both groups.

  9. A Dissecting Competition for Medical Students

    ERIC Educational Resources Information Center

    Samalia, Latika; Stringer, Mark D.

    2012-01-01

    After repeated requests from medical students for more cadaver dissection opportunities, a voluntary dissecting "competition" was initiated for the third year medical students in 2006. This has been held annually on five occasions since, offering up to 30 dissection stations and accommodating an average of 53 students (range 40-66) per year,…

  10. Comprehensive Analysis of the Membrane Phosphoproteome Regulated by Oligogalacturonides in Arabidopsis thaliana

    PubMed Central

    Mattei, Benedetta; Spinelli, Francesco; Pontiggia, Daniela; De Lorenzo, Giulia

    2016-01-01

    Early changes in the Arabidopsis thaliana membrane phosphoproteome in response to oligogalacturonides (OGs), a class of plant damage-associated molecular patterns (DAMPs), were analyzed by two complementary proteomic approaches. Differentially phosphorylated sites were determined through phosphopeptide enrichment followed by LC-MS/MS using label-free quantification; differentially phosphorylated proteins were identified by 2D-DIGE combined with phospho-specific fluorescent staining (phospho-DIGE). This large-scale phosphoproteome analysis of early OG-signaling enabled us to determine 100 regulated phosphosites using LC-MS/MS and 46 differential spots corresponding to 34 pdhosphoproteins using phospho-DIGE. Functional classification showed that the OG-responsive phosphoproteins include kinases, phosphatases and receptor-like kinases, heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, proteins related to cellular trafficking, transport, defense and signaling as well as novel candidates for a role in immunity, for which elicitor-induced phosphorylation changes have not been shown before. A comparison with previously identified elicitor-regulated phosphosites shows only a very limited overlap, uncovering the immune-related regulation of 70 phosphorylation sites and revealing novel potential players in the regulation of elicitor-dependent immunity. PMID:27532006

  11. HOPE-fixation of lung tissue allows retrospective proteome and phosphoproteome studies.

    PubMed

    Shevchuk, Olga; Abidi, Nada; Klawonn, Frank; Wissing, Josef; Nimtz, Manfred; Kugler, Christian; Steinert, Michael; Goldmann, Torsten; Jänsch, Lothar

    2014-11-07

    Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE)-fixation has been introduced as an alternative to formalin fixation of clinical samples. Beyond preservation of morphological structures for histology, HOPE-fixation was demonstrated to be compatible with recent methods for RNA and DNA sequencing. However, the suitability of HOPE-fixed materials for the inspection of proteomes by mass spectrometry so far remained undefined. This is of particular interest, since proteins constitute a prime resource for drug research and can give valuable insights into the activity status of signaling pathways. In this study, we extracted proteins from human lung tissue and tested HOPE-treated and snap-frozen tissues comparatively by proteome and phosphoproteome analyses. High confident data from accurate mass spectrometry allowed the identification of 2603 proteins and 3036 phosphorylation sites. HOPE-fixation did not hinder the representative extraction of proteins, and investigating their biochemical properties, covered subcellular localizations, and cellular processes revealed no bias caused by the type of fixation. In conclusion, proteome as well as phosphoproteome data of HOPE lung samples were qualitatively equivalent to results obtained from snap-frozen tissues. Thus, HOPE-treated tissues match clinical demands in both histology and retrospective proteome analyses of patient samples by proteomics.

  12. NeuCode Labeling in Nematodes: Proteomic and Phosphoproteomic Impact of Ascaroside Treatment in Caenorhabditis elegans*

    PubMed Central

    Rhoads, Timothy W.; Prasad, Aman; Kwiecien, Nicholas W.; Merrill, Anna E.; Zawack, Kelson; Westphall, Michael S.; Schroeder, Frank C.; Kimble, Judith; Coon, Joshua J.

    2015-01-01

    The nematode Caenorhabditis elegans is an important model organism for biomedical research. We previously described NeuCode stable isotope labeling by amino acids in cell culture (SILAC), a method for accurate proteome quantification with potential for multiplexing beyond the limits of traditional stable isotope labeling by amino acids in cell culture. Here we apply NeuCode SILAC to profile the proteomic and phosphoproteomic response of C. elegans to two potent members of the ascaroside family of nematode pheromones. By consuming labeled E. coli as part of their diet, C. elegans nematodes quickly and easily incorporate the NeuCode heavy lysine isotopologues by the young adult stage. Using this approach, we report, at high confidence, one of the largest proteomic and phosphoproteomic data sets to date in C. elegans: 6596 proteins at a false discovery rate ≤ 1% and 6620 phosphorylation isoforms with localization probability ≥75%. Our data reveal a post-translational signature of pheromone sensing that includes many conserved proteins implicated in longevity and response to stress. PMID:26392051

  13. NeuCode Labeling in Nematodes: Proteomic and Phosphoproteomic Impact of Ascaroside Treatment in Caenorhabditis elegans.

    PubMed

    Rhoads, Timothy W; Prasad, Aman; Kwiecien, Nicholas W; Merrill, Anna E; Zawack, Kelson; Westphall, Michael S; Schroeder, Frank C; Kimble, Judith; Coon, Joshua J

    2015-11-01

    The nematode Caenorhabditis elegans is an important model organism for biomedical research. We previously described NeuCode stable isotope labeling by amino acids in cell culture (SILAC), a method for accurate proteome quantification with potential for multiplexing beyond the limits of traditional stable isotope labeling by amino acids in cell culture. Here we apply NeuCode SILAC to profile the proteomic and phosphoproteomic response of C. elegans to two potent members of the ascaroside family of nematode pheromones. By consuming labeled E. coli as part of their diet, C. elegans nematodes quickly and easily incorporate the NeuCode heavy lysine isotopologues by the young adult stage. Using this approach, we report, at high confidence, one of the largest proteomic and phosphoproteomic data sets to date in C. elegans: 6596 proteins at a false discovery rate ≤ 1% and 6620 phosphorylation isoforms with localization probability ≥75%. Our data reveal a post-translational signature of pheromone sensing that includes many conserved proteins implicated in longevity and response to stress.

  14. Phosphoproteomic differences in major depressive disorder postmortem brains indicate effects on synaptic function.

    PubMed

    Martins-de-Souza, Daniel; Guest, Paul C; Vanattou-Saifoudine, Natacha; Rahmoune, Hassan; Bahn, Sabine

    2012-12-01

    There is still a lack in the molecular comprehension of major depressive disorder (MDD) although this condition affects approximately 10% of the world population. Protein phosphorylation is a posttranslational modification that regulates approximately one-third of the human proteins involved in a range of cellular and biological processes such as cellular signaling. Whereas phosphoproteome studies have been carried out extensively in cancer research, few such investigations have been carried out in studies of psychiatric disorders. Here, we present a comparative phosphoproteome analysis of postmortem dorsolateral prefrontal cortex tissues from 24 MDD patients and 12 control donors. Tissue extracts were analyzed using liquid chromatography mass spectrometry in a data-independent manner (LC-MS(E)). Our analyses resulted in the identification of 5,195 phosphopeptides, corresponding to 802 non-redundant proteins. Ninety of these proteins showed differential levels of phosphorylation in tissues from MDD subjects compared to controls, being 20 differentially phosphorylated in at least 2 peptides. The majority of these phosphorylated proteins were associated with synaptic transmission and cellular architecture not only pointing out potential biomarker candidates but mainly shedding light to the comprehension of MDD pathobiology.

  15. Large-Scale Phosphoproteomics Analysis of Whole Saliva Reveals a Distinct Phosphorylation Pattern

    PubMed Central

    Stone, Matthew D.; Chen, Xiaobing; McGowan, Thomas; Bandhakavi, Sricharan; Cheng, Bin; Rhodus, Nelson L.; Griffin, Timothy J.

    2011-01-01

    In-depth knowledge of bodily fluid phosphoproteomes, such as whole saliva, is limited. To better understand the whole saliva phosphoproteome, we generated a large-scale catalog of phosphorylated proteins. To circumvent the wide dynamic range of phosphoprotein abundance in whole saliva, we combined dynamic range compression using hexapeptide beads, strong cation exchange HPLC peptide fractionation, and immobilized metal affinity chromatography prior to mass spectrometry. In total, 217 unique phosphopeptides sites were identified representing 85 distinct phosphoproteins at 2.3% global FDR. From these peptides, 129 distinct phosphorylation sites were identified of which 57 were previously known, but only 11 of which had been previously identified in whole saliva. Cellular localization analysis revealed salivary phosphoproteins had a distribution similar to all known salivary proteins, but with less relative representation in “extracellular” and “plasma membrane” categories compared to salivary glycoproteins. Sequence alignment showed that phosphorylation occurred at acidic-directed kinase, proline-directed, and basophilic motifs. This differs from plasma phosphoproteins, which predominantly occur at Golgi casein kinase recognized sequences. Collectively, these results suggest diverse functions for salivary phosphoproteins and multiple kinases involved in their processing and secretion. In all, this study should lay groundwork for future elucidation of the functions of salivary protein phosphorylation. PMID:21299198

  16. Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.

    PubMed

    McNulty, Dean E; Annan, Roland S

    2008-05-01

    The diversity and complexity of proteins and peptides in biological systems requires powerful liquid chromatography-based separations to optimize resolution and detection of components. Proteomics strategies often combine two orthogonal separation modes to meet this challenge. In nearly all cases, the second dimension is a reverse phase separation interfaced directly to a mass spectrometer. Here we report on the use of hydrophilic interaction chromatography (HILIC) as part of a multidimensional chromatography strategy for proteomics. Tryptic peptides are separated on TSKgel Amide-80 columns using a shallow inverse organic gradient. Under these conditions, peptide retention is based on overall hydrophilicity, and a separation truly orthogonal to reverse phase is produced. Analysis of tryptic digests from HeLa cells yielded numbers of protein identifications comparable to that obtained using strong cation exchange. We also demonstrate that HILIC represents a significant advance in phosphoproteomics analysis. We exploited the strong hydrophilicity of the phosphate group to selectively enrich and fractionate phosphopeptides based on their increased retention under HILIC conditions. Subsequent IMAC enrichment of phosphopeptides from HILIC fractions showed better than 99% selectivity. This was achieved without the use of derivatization or chemical modifiers. In a 300-microg equivalent of HeLa cell lysate we identified over 1000 unique phosphorylation sites. More than 700 novel sites were added to the HeLa phosphoproteome.

  17. The phosphoproteome of Fusarium graminearum at the onset of nitrogen starvation.

    PubMed

    Rampitsch, Christof; Subramaniam, Rajagopal; Djuric-Ciganovic, Slavica; Bykova, Natalia V

    2010-01-01

    Fusarium graminearum grown under stress, such as nutrient deprivation, activates, among others, the trichothecene pathway that produces the mycotoxin deoxynivalenol and its derivatives. The kinase inhibitor staurosporine reduced the production of trichothecenes by 39% compared with control in vitro. On the other hand, phosphatase inhibitor okadaic acid increased the amount by 72% compared with the control in vitro. This suggests that phosphorylation events are involved in the signalling pathway, leading to the activation of the trichothecene pathway. Three approaches were used to study the phosphoproteome of F. graminearum under nitrogen-limiting conditions: 2-DE (2-DE: IEFxSDS-PAGE) in combination with MS protein identification; SDS-PAGE in combination with off-line IMAC and TiO(2) enrichment and gel electrophoresis LC-MS analysis; and a gel-free approach using strong anion exchange chromatography, IMAC and LC-MS. A total of 348 phosphorylation sites localized in 301 peptides from 241 proteins were identified. By 2-DE, 20 phosphoproteins were identified, nine of which underwent changes during the time course examined. Using gel electrophoresis LC-MS 231 phosphopeptides were identified from three samples (ten gel slices each) at time points of nitrogen starvation t=0, 6, and 12 h. The gel-free analysis added 70 peptides from 65 proteins to the total. Proteins of unknown function and enzymes of known function comprised the largest groups overall. Ten protein kinases and seven transcription factors were identified. This is the first reported phosphoproteome of F. graminearum.

  18. Analysis of dissection algorithms for vector computers

    NASA Technical Reports Server (NTRS)

    George, A.; Poole, W. G., Jr.; Voigt, R. G.

    1978-01-01

    Recently two dissection algorithms (one-way and incomplete nested dissection) have been developed for solving the sparse positive definite linear systems arising from n by n grid problems. Concurrently, vector computers (such as the CDC STAR-100 and TI ASC) have been developed for large scientific applications. An analysis of the use of dissection algorithms on vector computers dictates that vectors of maximum length be utilized thereby implying little or no dissection; on the other hand, minimizing operation counts suggest that considerable dissection be performed. In this paper we discuss the resolution of this conflict by minimizing the total time required by vectorized versions of the two algorithms.

  19. Analysis of the Rana catesbeiana tadpole tail fin proteome and phosphoproteome during T3-induced apoptosis: identification of a novel type I keratin

    PubMed Central

    Domanski, Dominik; Helbing, Caren C

    2007-01-01

    Background Thyroid hormones (THs) are vital in the maintenance of homeostasis and in the control of development. One postembryonic developmental process that is principally regulated by THs is amphibian metamorphosis. This process has been intensively studied at the genomic level yet very little information at the proteomic level exists. In addition, there is increasing evidence that changes in the phosphoproteome influence TH action. Results Here we identify components of the proteome and phosphoproteome in the tail fin that changed within 48 h of exposure of premetamorphic Rana catesbeiana tadpoles to 10 nM 3,5,3'-triiodothyronine (T3). To this end, we developed a cell and protein fractionation method combined with two-dimensional gel electrophoresis and phosphoprotein-specific staining. Altered proteins were identified using mass spectrometry (MS). We identified and cloned a novel Rana larval type I keratin, RLK I, which may be a target for caspase-mediated proteolysis upon exposure to T3. In addition, the RLK I transcript is reduced during T3-induced and natural metamorphosis which is consistent with a larval keratin. Furthermore, GILT, a protein involved in the immune system, is changed in phosphorylation state which is linked to its activation. Using a complementary MS technique for the analysis of differentially-expressed proteins, isobaric tags for relative and absolute quantitation (iTRAQ) revealed 15 additional proteins whose levels were altered upon T3 treatment. The success of identifying proteins whose levels changed upon T3 treatment with iTRAQ was enhanced through de novo sequencing of MS data and homology database searching. These proteins are involved in apoptosis, extracellular matrix structure, immune system, metabolism, mechanical function, and oxygen transport. Conclusion We have demonstrated the ability to derive proteomics-based information from a model species for postembryonic development for which no genome information is currently

  20. Novel aspects of grapevine response to phytoplasma infection investigated by a proteomic and phospho-proteomic approach with data integration into functional networks

    PubMed Central

    2013-01-01

    Background Translational and post-translational protein modifications play a key role in the response of plants to pathogen infection. Among the latter, phosphorylation is critical in modulating protein structure, localization and interaction with other partners. In this work, we used a multiplex staining approach with 2D gels to study quantitative changes in the proteome and phosphoproteome of Flavescence dorée-affected and recovered ‘Barbera’ grapevines, compared to healthy plants. Results We identified 48 proteins that differentially changed in abundance, phosphorylation, or both in response to Flavescence dorée phytoplasma infection. Most of them did not show any significant difference in recovered plants, which, by contrast, were characterized by changes in abundance, phosphorylation, or both for 17 proteins not detected in infected plants. Some enzymes involved in the antioxidant response that were up-regulated in infected plants, such as isocitrate dehydrogenase and glutathione S-transferase, returned to healthy-state levels in recovered plants. Others belonging to the same functional category were even down-regulated in recovered plants (oxidoreductase GLYR1 and ascorbate peroxidase). Our proteomic approach thus agreed with previously published biochemical and RT-qPCR data which reported down-regulation of scavenging enzymes and accumulation of H2O2 in recovered plants, possibly suggesting a role for this molecule in remission from infection. Fifteen differentially phosphorylated proteins (| ratio | > 2, p < 0.05) were identified in infected compared to healthy plants, including proteins involved in photosynthesis, response to stress and the antioxidant system. Many were not differentially phosphorylated in recovered compared to healthy plants, pointing to their specific role in responding to infection, followed by a return to a steady-state phosphorylation level after remission of symptoms. Gene ontology (GO) enrichment and statistical

  1. Phosphoproteomics Reveals HMGA1, a CK2 Substrate, as a Drug-Resistant Target in Non-Small Cell Lung Cancer

    PubMed Central

    Wang, Yi-Ting; Pan, Szu-Hua; Tsai, Chia-Feng; Kuo, Ting-Chun; Hsu, Yuan-Ling; Yen, Hsin-Yung; Choong, Wai-Kok; Wu, Hsin-Yi; Liao, Yen-Chen; Hong, Tse-Ming; Sung, Ting-Yi; Yang, Pan-Chyr; Chen, Yu-Ju

    2017-01-01

    Although EGFR tyrosine kinase inhibitors (TKIs) have demonstrated good efficacy in non-small-cell lung cancer (NSCLC) patients harboring EGFR mutations, most patients develop intrinsic and acquired resistance. We quantitatively profiled the phosphoproteome and proteome of drug-sensitive and drug-resistant NSCLC cells under gefitinib treatment. The construction of a dose-dependent responsive kinase-substrate network of 1548 phosphoproteins and 3834 proteins revealed CK2-centric modules as the dominant core network for the potential gefitinib resistance-associated proteins. CK2 knockdown decreased cell survival in gefitinib-resistant NSCLCs. Using motif analysis to identify the CK2 core sub-network, we verified that elevated phosphorylation level of a CK2 substrate, HMGA1 was a critical node contributing to EGFR-TKI resistance in NSCLC cell. Both HMGA1 knockdown or mutation of the CK2 phosphorylation site, S102, of HMGA1 reinforced the efficacy of gefitinib in resistant NSCLC cells through reactivation of the downstream signaling of EGFR. Our results delineate the TKI resistance-associated kinase-substrate network, suggesting a potential therapeutic strategy for overcoming TKI-induced resistance in NSCLC. PMID:28290473

  2. Unraveling the phosphoproteome dynamics in mammal mitochondria from a network perspective.

    PubMed

    Padrão, Ana Isabel; Vitorino, Rui; Duarte, José Alberto; Ferreira, Rita; Amado, Francisco

    2013-10-04

    With mitochondrion garnering more attention for its inextricable involvement in pathophysiological conditions, it seems imperative to understand the means by which the molecular pathways harbored in this organelle are regulated. Protein phosphorylation has been considered a central event in cellular signaling and, more recently, in the modulation of mitochondrial activity. Efforts have been made to understand the molecular mechanisms by which protein phosphorylation regulates mitochondrial signaling. With the advances in mass-spectrometry-based proteomics, there is a substantial hope and expectation in the increased knowledge of protein phosphorylation profile and its mode of regulation. On the basis of phosphorylation profiles, attempts have been made to disclose the kinases involved and how they control the molecular processes in mitochondria and, consequently, the cellular outcomes. Still, few studies have focused on mitochondrial phosphoproteome profiling, particularly in diseases. The present study reviews current data on protein phosphorylation profiling in mitochondria, the potential kinases involved and how pathophysiological conditions modulate the mitochondrial phosphoproteome. To integrate data from distinct research papers, we performed network analysis, with bioinformatic tools like Cytoscape, String, and PANTHER taking into consideration variables such as tissue specificity, biological processes, molecular functions, and pathophysiological conditions. For instance, data retrieved from these analyses evidence some homology in the mitochondrial phosphoproteome among liver and heart, with proteins from transport and oxidative phosphorylation clusters particularly susceptible to phosphorylation. A distinct profile was noticed for adipocytes, with proteins form metabolic processes, namely, triglycerides metabolism, as the main targets of phosphorylation. Regarding disease conditions, more phosphorylated proteins were observed in diabetics with some

  3. Genetic dissection of acetic acid tolerance in Saccharomyces cerevisiae.

    PubMed

    Geng, Peng; Xiao, Yin; Hu, Yun; Sun, Haiye; Xue, Wei; Zhang, Liang; Shi, Gui-Yang

    2016-09-01

    Dissection of the hereditary architecture underlying Saccharomyces cerevisiae tolerance to acetic acid is essential for ethanol fermentation. In this work, a genomics approach was used to dissect hereditary variations in acetic acid tolerance between two phenotypically different strains. A total of 160 segregants derived from these two strains were obtained. Phenotypic analysis indicated that the acetic acid tolerance displayed a normal distribution in these segregants, and suggested that the acetic acid tolerant traits were controlled by multiple quantitative trait loci (QTLs). Thus, 220 SSR markers covering the whole genome were used to detect QTLs of acetic acid tolerant traits. As a result, three QTLs were located on chromosomes 9, 12, and 16, respectively, which explained 38.8-65.9 % of the range of phenotypic variation. Furthermore, twelve genes of the candidates fell into the three QTL regions by integrating the QTL analysis with candidates of acetic acid tolerant genes. These results provided a novel avenue to obtain more robust strains.

  4. Acetylome and phosphoproteome modifications in imatinib resistant chronic myeloid leukaemia cells treated with valproic acid.

    PubMed

    Buchi, Francesca; Pastorelli, Roberta; Ferrari, Germano; Spinelli, Elena; Gozzini, Antonella; Sassolini, Francesca; Bosi, Alberto; Tombaccini, Donatella; Santini, Valeria

    2011-07-01

    Chronic myeloid leukaemia has a specific therapy: BCR/ABL inhibitor imatinib. Resistance due to BCR/ABL dependent and independent mechanisms is partially reversible by histone deacetylase inhibitors. We analysed by 2D-electrophoresis and anti-pan-acetylated and anti-phosphotyrosine immunoblots, followed by spot-matching and MALDI-TOF mass spectrometry, which proteome modifications would parallel restoration of sensitivity to imatinib by valproic acid (VPA). VPA plus imatinib significantly increased acetylation of HSP90 and hnRNP L and decreased phosphorylation of HSPs and hnRNPs in imatinib resistant cells. VPA was able to modify profoundly acetylome and phosphoproteome of CML cells, while reverting resistance to imatinib.

  5. Characterization of the human plasma phosphoproteome using linear ion trap mass spectrometry and multiple search engines.

    PubMed

    Carrascal, Montserrat; Gay, Marina; Ovelleiro, David; Casas, Vanessa; Gelpí, Emilio; Abian, Joaquin

    2010-02-05

    Major plasma protein families play different roles in blood physiology and hemostasis and in immunodefense. Other proteins in plasma can be involved in signaling as chemical messengers or constitute biological markers of the status of distant tissues. In this respect, the plasma phosphoproteome holds potentially relevant information on the mechanisms modulating these processes through the regulation of protein activity. In this work we describe for the first time a collection of phosphopeptides identified in human plasma using immunoaffinity separation of the seven major serum protein families from other plasma proteins, SCX fractionation, and TiO(2) purification prior to LC-MS/MS analysis. One-hundred and twenty-seven phosphosites in 138 phosphopeptides mapping 70 phosphoproteins were identified with FDR < 1%. A high-confidence collection of phosphosites was obtained using a combined search with the OMSSA, SEQUEST, and Phenyx search engines.

  6. Phosphoproteomics as an emerging weapon to develop new antibiotics against carbapenem resistant strain of Acinetobacter baumannii.

    PubMed

    Tiwari, Vishvanath; Tiwari, Monalisa

    2015-01-01

    Acinetobacter baumannii causes pneumonia, bloodstream infections, urinary tract infections, respiratory infections and meningitis. A. baumannii has developed resistance against most of the antibiotics including carbapenem. Therefore, to battle carbapenem resistance, there is a need to develop antimicrobial drugs with new modes of action. Phosphoproteomics will help identify the differentially phosphorylated protein and its crucial phosphosites which facilitate the elucidation of molecular mechanism of signaling and regulation of carbapenem resistant strain of A. baumannii as compared to carbapenem sensitive strain. This understanding might be useful for the development of new antibiotics against kinases involved in the phosphorylation of identified phosphosites in carbapenem resistant strain of A. baumannii. The proposed antibiotics selectively inhibit carbapenem resistant strain which further avoids its excessive use against carbapenem sensitive strain and thereafter reduces emergence of resistance.

  7. Mitochondrial tyrosine phosphoproteome: new insights from an up-to-date analysis.

    PubMed

    Cesaro, Luca; Salvi, Mauro

    2010-01-01

    Tyrosine phosphorylation is a newcomer in the mitochondrial signaling and is currently emerging as an important mechanism for regulating mitochondrial processes. But to what extent? By analyzing an updated draft of the mitochondrial tyrosine phosphoproteome, the following observations can be drawn: more than a hundred mitochondrial proteins undergo tyrosine phosphorylation, phosphotyrosine proteins are distributed in each of the submitochondrial compartments, and mitochondrial tyrosine phosphorylated proteins are involved in a variety of functions as metabolism (electron transport chain, Krebs cycle, fatty acid and amino acid metabolism), solute and protein transport, mitochondrial translation machinery, quality protein assessment, oxidative stress, apoptosis, fission, and other. This large and varied collection suggests that tyrosine phosphorylation could be a widespread mechanism in modulating mitochondrial functions. Moreover the in silico model is here used to explore potential effects of tyrosine phosphorylation on selected mitochondrial proteins pointing out some future perspectives in this field.

  8. Automated Immobilized Metal Affinity Chromatography System for Enrichment of Escherichia coli Phosphoproteome

    SciTech Connect

    Qu, Yi; Wu, Si; Zhao, Rui; Zink, Erika M.; Orton, Daniel J.; Moore, Ronald J.; Meng, Da; Clauss, Therese RW; Aldrich, Joshua T.; Lipton, Mary S.; Pasa-Tolic, Ljiljana

    2013-06-05

    Enrichment of bacterial phosphopeptides is an essential step prior to bottom-up mass spectrometry-based analysis of the phosphoproteome, which is fundamental to understanding the role of phosphoproteins in cell signaling and regulation of protein activity. We developed an automated IMAC system to enrich strong cation exchange-fractionated phosphopeptides from the soluble proteome of Escherichia coli MG1655 grown on minimal medium. Initial demonstration of the system resulted in identification of 75 phosphopeptides covering 52 phosphoproteins. Consistent with previous studies, many of these phosphoproteins are involved in the carbohydrate portion of central metabolism. The automated system utilizes a large capacity IMAC column that can effectively enrich phosphopeptides from a bacterial sample by increasing peptide loading and reducing the wash time. An additional benefit of the automated IMAC system is reduced labor and associated costs.

  9. Celiac Anti-Type 2 Transglutaminase Antibodies Induce Phosphoproteome Modification in Intestinal Epithelial Caco-2 Cells

    PubMed Central

    Marabotti, Anna; Lepretti, Marilena; Salzano, Anna Maria; Scaloni, Andrea; Vitale, Monica; Zambrano, Nicola; Sblattero, Daniele; Esposito, Carla

    2013-01-01

    Background Celiac disease is an inflammatory condition of the small intestine that affects genetically predisposed individuals after dietary wheat gliadin ingestion. Type 2-transglutaminase (TG2) activity seems to be responsible for a strong autoimmune response in celiac disease, TG2 being the main autoantigen. Several studies support the concept that celiac anti-TG2 antibodies may contribute to disease pathogenesis. Our recent findings on the ability of anti-TG2 antibodies to induce a rapid intracellular mobilization of calcium ions, as well as extracellular signal-regulated kinase phosphorylation, suggest that they potentially act as signaling molecules. In line with this concept, we have investigated whether anti-TG2 antibodies can induce phosphoproteome modification in an intestinal epithelial cell line. Methods and Principal Findings We studied phosphoproteome modification in Caco-2 cells treated with recombinant celiac anti-TG2 antibodies. We performed a two-dimensional electrophoresis followed by specific staining of phosphoproteins and mass spectrometry analysis of differentially phosphorylated proteins. Of 14 identified proteins (excluding two uncharacterized proteins), three were hypophosphorylated and nine were hyperphosphorylated. Bioinformatics analyses confirmed the presence of phosphorylation sites in all the identified proteins and highlighted their involvement in several fundamental biological processes, such as cell cycle progression, cell stress response, cytoskeletal organization and apoptosis. Conclusions Identification of differentially phosphorylated proteins downstream of TG2-antibody stimulation suggests that in Caco-2 cells these antibodies perturb cell homeostasis by behaving as signaling molecules. We hypothesize that anti-TG2 autoantibodies may destabilize the integrity of the intestinal mucosa in celiac individuals, thus contributing to celiac disease establishment and progression. Since several proteins here identified in this study

  10. Phosphoproteomic profiling of human myocardial tissues distinguishes ischemic from non-ischemic end stage heart failure.

    PubMed

    Schechter, Matthew A; Hsieh, Michael K H; Njoroge, Linda W; Thompson, J Will; Soderblom, Erik J; Feger, Bryan J; Troupes, Constantine D; Hershberger, Kathleen A; Ilkayeva, Olga R; Nagel, Whitney L; Landinez, Gina P; Shah, Kishan M; Burns, Virginia A; Santacruz, Lucia; Hirschey, Matthew D; Foster, Matthew W; Milano, Carmelo A; Moseley, M Arthur; Piacentino, Valentino; Bowles, Dawn E

    2014-01-01

    The molecular differences between ischemic (IF) and non-ischemic (NIF) heart failure are poorly defined. A better understanding of the molecular differences between these two heart failure etiologies may lead to the development of more effective heart failure therapeutics. In this study extensive proteomic and phosphoproteomic profiles of myocardial tissue from patients diagnosed with IF or NIF were assembled and compared. Proteins extracted from left ventricular sections were proteolyzed and phosphopeptides were enriched using titanium dioxide resin. Gel- and label-free nanoscale capillary liquid chromatography coupled to high resolution accuracy mass tandem mass spectrometry allowed for the quantification of 4,436 peptides (corresponding to 450 proteins) and 823 phosphopeptides (corresponding to 400 proteins) from the unenriched and phospho-enriched fractions, respectively. Protein abundance did not distinguish NIF from IF. In contrast, 37 peptides (corresponding to 26 proteins) exhibited a ≥ 2-fold alteration in phosphorylation state (p<0.05) when comparing IF and NIF. The degree of protein phosphorylation at these 37 sites was specifically dependent upon the heart failure etiology examined. Proteins exhibiting phosphorylation alterations were grouped into functional categories: transcriptional activation/RNA processing; cytoskeleton structure/function; molecular chaperones; cell adhesion/signaling; apoptosis; and energetic/metabolism. Phosphoproteomic analysis demonstrated profound post-translational differences in proteins that are involved in multiple cellular processes between different heart failure phenotypes. Understanding the roles these phosphorylation alterations play in the development of NIF and IF has the potential to generate etiology-specific heart failure therapeutics, which could be more effective than current therapeutics in addressing the growing concern of heart failure.

  11. Deciphering the acute cellular phosphoproteome response to irradiation with X-rays, protons and carbon ions.

    PubMed

    Winter, Martin; Dokic, Ivana; Schlegel, Julian; Warnken, Uwe; Debus, Jürgen; Abdollahi, Amir; Schnölzer, Martina

    2017-03-16

    Radiotherapy is a cornerstone of cancer therapy. The recently established particle therapy with raster-scanning protons and carbon ions landmarks a new era in the field of high-precision cancer medicine. However, molecular mechanisms governing radiation induced intracellular signaling remain elusive. Here, we present the first comprehensive proteomic and phosphoproteomic study applying stable isotope labeling by amino acids in cell culture (SILAC) in combination with high-resolution mass spectrometry to decipher cellular response to irradiation with X-rays, protons and carbon ions. At protein expression level limited alterations were observed 2h post irradiation of human lung adenocarcinoma cells. In contrast, 181 phosphorylation sites were found to be differentially regulated out of which 151 sites were not hitherto attributed to radiation response as revealed by crosscheck with the PhosphoSitePlus database. Radiation-induced phosphorylation of the p(S/T)Q motif was the prevailing regulation pattern affecting proteins involved in DNA damage response signaling. Since radiation doses were selected to produce same level of cell kill and DNA double-strand breakage for each radiation quality, DNA damage responsive phosphorylation sites were regulated to same extent. However, differential phosphorylation between radiation qualities was observed for 55 phosphorylation sites indicating the existence of distinct signaling circuitries induced by X-ray versus particle (proton/carbon) irradiation beyond the canonical DNA damage response. This unexpected finding was confirmed in targeted spike-in experiments using synthetic isotope labeled phosphopeptides. Herewith, we successfully validated uniform DNA damage response signaling coexisting with altered signaling involved in apoptosis and metabolic processes induced by X-ray and particle based treatments. In summary, the comprehensive insight into the radiation-induced phosphoproteome landscape is instructive for the design of

  12. Phosphoproteomic Profiling of Human Myocardial Tissues Distinguishes Ischemic from Non-Ischemic End Stage Heart Failure

    PubMed Central

    Njoroge, Linda W.; Thompson, J. Will; Soderblom, Erik J.; Feger, Bryan J.; Troupes, Constantine D.; Hershberger, Kathleen A.; Ilkayeva, Olga R.; Nagel, Whitney L.; Landinez, Gina P.; Shah, Kishan M.; Burns, Virginia A.; Santacruz, Lucia; Hirschey, Matthew D.; Foster, Matthew W.; Milano, Carmelo A.; Moseley, M. Arthur; Piacentino, Valentino; Bowles, Dawn E.

    2014-01-01

    The molecular differences between ischemic (IF) and non-ischemic (NIF) heart failure are poorly defined. A better understanding of the molecular differences between these two heart failure etiologies may lead to the development of more effective heart failure therapeutics. In this study extensive proteomic and phosphoproteomic profiles of myocardial tissue from patients diagnosed with IF or NIF were assembled and compared. Proteins extracted from left ventricular sections were proteolyzed and phosphopeptides were enriched using titanium dioxide resin. Gel- and label-free nanoscale capillary liquid chromatography coupled to high resolution accuracy mass tandem mass spectrometry allowed for the quantification of 4,436 peptides (corresponding to 450 proteins) and 823 phosphopeptides (corresponding to 400 proteins) from the unenriched and phospho-enriched fractions, respectively. Protein abundance did not distinguish NIF from IF. In contrast, 37 peptides (corresponding to 26 proteins) exhibited a ≥2-fold alteration in phosphorylation state (p<0.05) when comparing IF and NIF. The degree of protein phosphorylation at these 37 sites was specifically dependent upon the heart failure etiology examined. Proteins exhibiting phosphorylation alterations were grouped into functional categories: transcriptional activation/RNA processing; cytoskeleton structure/function; molecular chaperones; cell adhesion/signaling; apoptosis; and energetic/metabolism. Phosphoproteomic analysis demonstrated profound post-translational differences in proteins that are involved in multiple cellular processes between different heart failure phenotypes. Understanding the roles these phosphorylation alterations play in the development of NIF and IF has the potential to generate etiology-specific heart failure therapeutics, which could be more effective than current therapeutics in addressing the growing concern of heart failure. PMID:25117565

  13. Systematic Analysis of the Phosphoproteome and Kinase-substrate Networks in the Mouse Testis*

    PubMed Central

    Qi, Lin; Liu, Zexian; Wang, Jing; Cui, Yiqiang; Guo, Yueshuai; Zhou, Tao; Zhou, Zuomin; Guo, Xuejiang; Xue, Yu; Sha, Jiahao

    2014-01-01

    Spermatogenesis is a complex process closely associated with the phosphorylation-orchestrated cell cycle. Elucidating the phosphorylation-based regulations should advance our understanding of the underlying molecular mechanisms. Here we present an integrative study of phosphorylation events in the testis. Large-scale phosphoproteome profiling in the adult mouse testis identified 17,829 phosphorylation sites in 3955 phosphoproteins. Although only approximately half of the phosphorylation sites enriched by IMAC were also captured by TiO2, both the phosphoprotein data sets identified by the two methods significantly enriched the functional annotation of spermatogenesis. Thus, the phosphoproteome profiled in this study is a highly useful snapshot of the phosphorylation events in spermatogenesis. To further understand phosphoregulation in the testis, the site-specific kinase-substrate relations were computationally predicted for reconstructing kinase-substrate phosphorylation networks. A core sub-kinase-substrate phosphorylation networks among the spermatogenesis-related proteins was retrieved and analyzed to explore the phosphoregulation during spermatogenesis. Moreover, network-based analyses demonstrated that a number of protein kinases such as MAPKs, CDK2, and CDC2 with statistically more site-specific kinase-substrate relations might have significantly higher activities and play an essential role in spermatogenesis, and the predictions were consistent with previous studies on the regulatory roles of these kinases. In particular, the analyses proposed that the activities of POLO-like kinases (PLKs) might be dramatically higher, while the prediction was experimentally validated by detecting and comparing the phosphorylation levels of pT210, an indicator of PLK1 activation, in testis and other tissues. Further experiments showed that the inhibition of POLO-like kinases decreases cell proliferation by inducing G2/M cell cycle arrest. Taken together, this systematic

  14. Phosphatase of Regenerating Liver 3 (PRL3) Provokes a Tyrosine Phosphoproteome to Drive Prometastatic Signal Transduction*

    PubMed Central

    Walls, Chad D.; Iliuk, Anton; Bai, Yunpeng; Wang, Mu; Tao, W. Andy; Zhang, Zhong-Yin

    2013-01-01

    Phosphatase of regenerating liver 3 (PRL3) is suspected to be a causative factor toward cellular metastasis when in excess. To date, the molecular basis for PRL3 function remains an enigma, making efforts at distilling a concerted mechanism for PRL3-mediated metastatic dissemination very difficult. We previously discovered that PRL3 expressing cells exhibit a pronounced increase in protein tyrosine phosphorylation. Here we take an unbiased mass spectrometry-based approach toward identifying the phosphoproteins exhibiting enhanced levels of tyrosine phosphorylation with a goal to define the “PRL3-mediated signaling network.” Phosphoproteomic data support intracellular activation of an extensive signaling network normally governed by extracellular ligand-activated transmembrane growth factor, cytokine, and integrin receptors in the PRL3 cells. Additionally, data implicate the Src tyrosine kinase as the major intracellular kinase responsible for “hijacking” this network and provide strong evidence that aberrant Src activation is a major consequence of PRL3 overexpression. Importantly, the data support a PDGF(α/β)-, Eph (A2/B3/B4)-, and Integrin (β1/β5)-receptor array as being the predominant network coordinator in the PRL3 cells, corroborating a PRL3-induced mesenchymal-state. Within this network, we find that tyrosine phosphorylation is increased on a multitude of signaling effectors responsible for Rho-family GTPase, PI3K-Akt, STAT, and ERK activation, linking observations made by the field as a whole under Src as a primary signal transducer. Our phosphoproteomic data paint the most comprehensive picture to date of how PRL3 drives prometastatic molecular events through Src activation. PMID:24030100

  15. Phosphoproteomic analysis of interacting tumor and endothelial cells identifies regulatory mechanisms of transendothelial migration.

    PubMed

    Locard-Paulet, Marie; Lim, Lindsay; Veluscek, Giulia; McMahon, Kelly; Sinclair, John; van Weverwijk, Antoinette; Worboys, Jonathan D; Yuan, Yinyin; Isacke, Clare M; Jørgensen, Claus

    2016-02-09

    The exit of metastasizing tumor cells from the vasculature, extravasation, is regulated by their dynamic interactions with the endothelial cells that line the internal surface of vessels. To elucidate signals controlling tumor cell adhesion to the endothelium and subsequent transendothelial migration, we performed phosphoproteomic analysis to map cell-specific changes in protein phosphorylation that were triggered by contact between metastatic MDA-MB-231 breast cancer cells and endothelial cells. From the 2669 unique phosphorylation sites identified, 77 and 43 were differentially phosphorylated in the tumor cells and endothelial cells, respectively. The receptor tyrosine kinase ephrin type A receptor 2 (EPHA2) exhibited decreased Tyr(772) phosphorylation in the cancer cells upon endothelial contact. Knockdown of EPHA2 increased adhesion of the breast cancer cells to human umbilical vein endothelial cells (HUVECs) and their transendothelial migration in coculture cell assays, as well as early-stage lung colonization in vivo. EPHA2-mediated inhibition of transendothelial migration of breast cancer cells depended on interaction with the ligand ephrinA1 on HUVECs and phosphorylation of EPHA2-Tyr(772). When EPHA2 phosphorylation dynamics were compared between cell lines of different metastatic ability, EPHA2-Tyr(772) was rapidly dephosphorylated after ephrinA1 stimulation specifically in cells targeting the lung. Knockdown of the phosphatase LMW-PTP reduced adhesion and transendothelial migration of the breast cancer cells. Overall, cell-specific phosphoproteomic analysis provides a bidirectional map of contact-initiated signaling between tumor and endothelial cells that can be further investigated to identify mechanisms controlling the transendothelial cell migration of cancer cells.

  16. Evaluation of Educator & Student Use of & Attitudes toward Dissection & Dissection Alternatives

    ERIC Educational Resources Information Center

    Osenkowski, Pamela; Green, Che; Tjaden, Anne; Cunniff, Peggy

    2015-01-01

    Animal dissection has been routinely practiced in American biology classrooms for decades. With technological advancements, more states adopting student choice measures, and increased awareness about ethical concerns surrounding dissection, many useful dissection alternatives have been developed. To understand the current use of animal dissection…

  17. Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia

    PubMed Central

    Hernandez-Valladares, Maria; Aasebø, Elise; Selheim, Frode; Berven, Frode S.; Bruserud, Øystein

    2016-01-01

    Global mass spectrometry (MS)-based proteomic and phosphoproteomic studies of acute myeloid leukemia (AML) biomarkers represent a powerful strategy to identify and confirm proteins and their phosphorylated modifications that could be applied in diagnosis and prognosis, as a support for individual treatment regimens and selection of patients for bone marrow transplant. MS-based studies require optimal and reproducible workflows that allow a satisfactory coverage of the proteome and its modifications. Preparation of samples for global MS analysis is a crucial step and it usually requires method testing, tuning and optimization. Different proteomic workflows that have been used to prepare AML patient samples for global MS analysis usually include a standard protein in-solution digestion procedure with a urea-based lysis buffer. The enrichment of phosphopeptides from AML patient samples has previously been carried out either with immobilized metal affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC). We have recently tested several methods of sample preparation for MS analysis of the AML proteome and phosphoproteome and introduced filter-aided sample preparation (FASP) as a superior methodology for the sensitive and reproducible generation of peptides from patient samples. FASP-prepared peptides can be further fractionated or IMAC-enriched for proteome or phosphoproteome analyses. Herein, we will review both in-solution and FASP-based sample preparation workflows and encourage the use of the latter for the highest protein and phosphorylation coverage and reproducibility. PMID:28248234

  18. Mass spectrometric phosphoproteome analysis of HIV-infected brain reveals novel phosphorylation sites and differential phosphorylation patterns

    PubMed Central

    Uzasci, Lerna; Auh, Sungyoung; Cotter, Robert J.; Nath, Avindra

    2016-01-01

    Purpose To map the phosphoproteome and identify changes in the phosphorylation patterns in the HIV-infected and uninfected brain using high-resolution mass spectrometry. Experimental Design Parietal cortex from brain of individuals with and without HIV infection were lysed and trypsinized. The peptides were labeled with iTRAQ reagents, combined, phospho-enriched by titanium dioxide chromatography, and analyzed by LC-MS/MS with high-resolution. Results Our phosphoproteomic workflow resulted in the identification of 112 phosphorylated proteins and 17 novel phosphorylation sites in all the samples that were analyzed. The phosphopeptide sequences were searched for kinase substrate motifs which revealed potential kinases involved in important signaling pathways. The site-specific phosphopeptide quantification showed that peptides from neurofilament medium polypeptide, myelin basic protein, and 2′–3′-cyclic nucleotide-3′ phosphodiesterase have relatively higher phosphorylation levels during HIV infection. Clinical Relevance This study has enriched the global phosphoproteome knowledge of the human brain by detecting novel phosphorylation sites on neuronal proteins and identifying differentially phosphorylated brain proteins during HIV infection. Kinases that lead to unusual phosphorylations could be therapeutic targets for the treatment of HIV-associated neurocognitive disorders (HAND). PMID:26033855

  19. Comparative Phosphoproteomic Analysis under High-Nitrogen Fertilizer Reveals Central Phosphoproteins Promoting Wheat Grain Starch and Protein Synthesis

    PubMed Central

    Zhen, Shoumin; Deng, Xiong; Zhang, Ming; Zhu, Gengrui; Lv, Dongwen; Wang, Yaping; Zhu, Dong; Yan, Yueming

    2017-01-01

    Nitrogen (N) is a macronutrient important for plant growth and development. It also strongly influences starch and protein synthesis, closely related to grain yield and quality. We performed the first comparative phosphoproteomic analysis of developing wheat grains in response to high-N fertilizer. Physiological and biochemical analyses showed that application of high-N fertilizer resulted in significant increases in leaf length and area, chlorophyll content, the activity of key enzymes in leaves such as nitrate reductase (NR), and in grains such as sucrose phosphate synthase (SPS), sucrose synthase (SuSy), and ADP glucose pyrophosphorylase (AGPase). This enhanced enzyme activity led to significant improvements in starch content, grain yield, and ultimately, bread making quality. Comparative phosphoproteomic analysis of developing grains under the application of high-N fertilizer performed 15 and 25 days post-anthesis identified 2470 phosphosites among 1372 phosphoproteins, of which 411 unique proteins displayed significant changes in phosphorylation level (>2-fold or <0.5-fold). These phosphoproteins are involved mainly in signaling transduction, starch synthesis, energy metabolism. Pro-Q diamond staining and Western blotting confirmed our phosphoproteomic results. We propose a putative pathway to elucidate the important roles of the central phosphoproteins regulating grain starch and protein synthesis. Our results provide new insights into the molecular mechanisms of protein phosphorylation modifications involved in grain development, yield and quality formation. PMID:28194157

  20. Keeping Dissection Alive for Medical Students

    ERIC Educational Resources Information Center

    Chambers, James; Emlyn-Jones, Daniel

    2009-01-01

    Traditional dissection teaching is being reduced in a number of medical schools, particularly in the United Kingdom. In response to this, 12 medical students from Warwick University, UK, traveled to the Island of Grenada for an intensive extracurricular dissection course at St. George's University. This course not only benefited the host…

  1. Quick Dissection of the Segmental Bronchi

    ERIC Educational Resources Information Center

    Nakajima, Yuji

    2010-01-01

    Knowledge of the three-dimensional anatomy of the bronchopulmonary segments is essential for respiratory medicine. This report describes a quick guide for dissecting the segmental bronchi in formaldehyde-fixed human material. All segmental bronchi are easy to dissect, and thus, this exercise will help medical students to better understand the…

  2. Aortic arch dissection: a controversy of classification.

    PubMed

    Lempel, Jason K; Frazier, Aletta Ann; Jeudy, Jean; Kligerman, Seth J; Schultz, Randall; Ninalowo, Hammed A; Gozansky, Elliott K; Griffith, Bartley; White, Charles S

    2014-06-01

    Aortic dissections originating in the ascending aorta and descending aorta have been classified as type A and type B dissections, respectively. However, dissections with intimal flap extension into the aortic arch between the innominate and left subclavian arteries are not accounted for adequately in the widely used Stanford classification. This gap has been the subject of controversy in the medical and surgical literature, and there is a tendency among many radiologists to categorize such arch dissections as type A lesions, thus making them an indication for surgery. However, the radiologic perspective is not supported by either standard dissection classification or current clinical management. In this special report, the origin of dissection classification and its evolution into current radiologic interpretation and surgical practice are reviewed. The cause for the widespread misconception about classification and treatment algorithms is identified. Institutional review board approval and waiver of informed consent were obtained as part of this HIPAA-compliant retrospective study to assess all aortic dissection studies performed at the University of Maryland Medical Center, Baltimore between 2010 and 2012 to determine the prevalence of arch dissections. Finally, a unified classification system that reconciles imaging interpretation and management implementation is proposed.

  3. [Internal carotid artery dissection after Heimlich maneuver].

    PubMed

    Rakotoharinandrasana, H; Petit, E; Dumas, P; Vandermarcq, P; Gil, R; Neau, J-Ph

    2003-01-01

    We report a case of cervical artery dissection following a Heimlich maneuver. Cervical artery dissections are at the present time well known and are sometimes associated with trivial traumas. However, to our knowledge, this complication of such maneuver was never reported in the literature. Pathophysiological mechanisms are discussed.

  4. Spontaneous Coronary Artery Dissection with Cardiac Tamponade.

    PubMed

    Goh, Anne C H; Lundstrom, Robert J

    2015-10-01

    Spontaneous coronary artery dissection is a rare cause of acute coronary syndrome. Clinical presentation ranges from chest pain alone to ST-segment-elevation myocardial infarction, ventricular fibrillation, and sudden death. The treatment of patients with spontaneous coronary artery dissection is challenging because the disease pathophysiology is unclear, optimal treatment is unknown, and short- and long-term prognostic data are minimal. We report the case of a 70-year-old woman who presented with an acute ST-segment-elevation myocardial infarction secondary to a spontaneous dissection of the left anterior descending coronary artery. She was treated conservatively. Cardiac tamponade developed 16 hours after presentation. Repeat coronary angiography revealed extension of the dissection. Medical therapy was continued after the hemopericardium was aspirated. The patient remained asymptomatic 3 years after hospital discharge. To our knowledge, this is the first reported case of spontaneous coronary artery dissection in association with cardiac tamponade that was treated conservatively and had a successful outcome.

  5. Spontaneous Coronary Artery Dissection during Cabergoline Therapy

    PubMed Central

    Mehta, Nishaki Kiran; Malkani, Samir; Ockene, Ira

    2012-01-01

    Although spontaneous coronary artery dissection is a rare cause of acute coronary syndrome, it should be considered during the evaluation of patients who have chest pain. Coronary vasospasm can lead to spontaneous dissection. The dopamine agonist cabergoline is known to cause digital vasospasm. Herein, we report a case of spontaneous right coronary artery dissection in a 43-year-old woman who was taking cabergoline as therapy for prolactinoma. To our knowledge, this is the first report of an apparent relationship between cabergoline therapy and spontaneous coronary artery dissection. The possible association of cabergoline with coronary artery spasm and dissection should be considered in patients who present with chest pain while taking this medication. PMID:22412238

  6. Acute aortic dissection at two extreme ages.

    PubMed

    Ramzisham, A R M; Arief, H; Ngoo, K S; Zamrin, D M; Joanna, O S M

    2011-01-01

    Acute aortic dissection is a life-threatening condition, warranting prompt diagnosis and treatment. Management of which incorporates multidisciplinary expertise from the medical, surgical and intensive care. If left untreated, the mortality rate of acute aortic disease exceeds 50% within 48 hours and 80% within two weeks, with a 5-year survival rate of 19%. The most common cause of death in untreated acute aortic dissection, regardless of aetiology, is aortic rupture. We would like to share our successful experience of cases at the two extreme ages of acute aortic dissection. Literature review with their pathogenesis are discussed.

  7. Identification of BCAP-{sub L} as a negative regulator of the TLR signaling-induced production of IL-6 and IL-10 in macrophages by tyrosine phosphoproteomics

    SciTech Connect

    Matsumura, Takayuki; Oyama, Masaaki; Kozuka-Hata, Hiroko; Ishikawa, Kosuke; Inoue, Takafumi; Muta, Tatsushi; Semba, Kentaro; Inoue, Jun-ichiro

    2010-09-17

    Research highlights: {yields} Twenty five tyrosine-phosphorylated proteins in LPS-stimulated macrophages were determined. {yields} BCAP is a novel tyrosine-phosphorylated protein in LPS-stimulated macrophages. {yields} BCAP-{sub L} inhibits IL-6 and IL-10 production in LPS-stimulated macrophages. -- Abstract: Toll-like receptor (TLR) signaling in macrophages is essential for anti-pathogen responses such as cytokine production and antigen presentation. Although numerous reports suggest that protein tyrosine kinases (PTKs) are involved in cytokine induction in response to lipopolysaccharides (LPS; TLR4 ligand) in macrophages, the PTK-mediated signal transduction pathway has yet to be analyzed in detail. Here, we carried out a comprehensive and quantitative dynamic tyrosine phosphoproteomic analysis on the TLR4-mediated host defense system in RAW264.7 macrophages using stable isotope labeling by amino acids in cell culture (SILAC). We determined the temporal profiles of 25 proteins based on SILAC-encoded peptide(s). Of these, we focused on the tyrosine phosphorylation of B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) because the function of BCAP remains unknown in TLR signaling in macrophages. Furthermore, Bcap has two distinct transcripts, a full-length (Bcap-{sub L}) and an alternatively initiated or spliced (Bcap-{sub S}) mRNA, and little is known about the differential functions of the BCAP-{sub L} and BCAP-{sub S} proteins. Our study showed, for the first time, that RNAi-mediated selective depletion of BCAP-{sub L} enhanced IL-6 and IL-10 production but not TNF-{alpha} production in TLR ligand-stimulated macrophages. We propose that BCAP-{sub L} (but not BCAP-{sub S}) is a negative regulator of the TLR-mediated host defense system in macrophages.

  8. Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants.

    PubMed

    Iwai, Leo K; Payne, Leo S; Luczynski, Maciej T; Chang, Francis; Xu, Huifang; Clinton, Ryan W; Paul, Angela; Esposito, Edward A; Gridley, Scott; Leitinger, Birgit; Naegle, Kristen M; Huang, Paul H

    2013-09-15

    Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens.

  9. Niobium(V) oxide (Nb2O5): application to phosphoproteomics.

    PubMed

    Ficarro, Scott B; Parikh, Jignesh R; Blank, Nathaniel C; Marto, Jarrod A

    2008-06-15

    Proteomics-based analysis of signaling cascades relies on a growing suite of affinity resins and methods aimed at efficient enrichment of phosphorylated peptides from complex biological mixtures. Given the heterogeneity of phosphopeptides and the overlap in chemical properties between phospho- and unmodified peptides, it is likely that the use of multiple resins will provide the best combination of specificity, yield, and coverage for large-scale proteomics studies. Recently titanium and zirconium dioxides have been used successfully for enrichment of phosphopeptides. Here we report the first demonstration that niobium pentoxide (Nb 2O 5) provides for efficient enrichment and recovery ( approximately 50-100%) of phosphopeptides from simple mixtures and facilitates identification of several hundred putative sites of phosphorylation from cell lysate. Comparison of phosphorylated peptides identified from Nb 2O 5 and TiO 2 with sequences in the PhosphoELM database suggests a useful degree of divergence in the selectivity of these metal oxide resins. Collectively our data indicate that Nb 2O 5 provides efficient enrichment for phosphopeptides and offers a complementary approach for large-scale phosphoproteomics studies.

  10. Phosphoproteomic Analysis of Paper Mulberry Reveals Phosphorylation Functions in Chilling Tolerance.

    PubMed

    Pi, Zhi; Zhao, Mei-Ling; Peng, Xian-Jun; Shen, Shi-Hua

    2017-04-13

    Paper mulberry is a valuable woody species with a good chilling tolerance. In this study, phosphoproteomic analysis, physiological measurement, and mRNA quantification were employed to explore the molecular mechanism of chilling (4 °C) tolerance in paper mulberry. After chilling for 6 h, 427 significantly changed phosphoproteins were detected in paper mulberry seedlings without obvious physiological injury. When obvious physiological injury occurred after chilling for 48 h, a total of 611 phosphoproteins were found to be significantly changed at the phosphorylation level. Several protein kinases, especially CKII, were possibly responsible for these changes according to conserved sequence analysis. The results of Gene Ontology analysis showed that phosphoproteins were mainly responsible for signal transduction, protein modification, and translation during chilling. Additionally, transport and cellular component organization were enriched after chilling for 6 and 48 h, respectively. On the basis of the protein-protein interaction network analysis, a protein kinase and phosphatases hub protein (P1959) were found to be involved in cross-talk between Ca(2+), BR, ABA, and ethylene-mediated signaling pathways. We also highlighted the phosphorylation of BpSIZ1 and BpICE1 possibly impacted on the CBF/DREB-responsive pathway. From these results, we developed a schematic for the chilling tolerance mechanism at phosphorylation level.

  11. Phosphoproteomic dynamics of chickpea (Cicer arietinum L.) reveals shared and distinct components of dehydration response.

    PubMed

    Subba, Pratigya; Barua, Pragya; Kumar, Rajiv; Datta, Asis; Soni, Kamlesh Kumar; Chakraborty, Subhra; Chakraborty, Niranjan

    2013-11-01

    Reversible protein phosphorylation is a ubiquitous regulatory mechanism that plays critical roles in transducing stress signals to bring about coordinated intracellular responses. To gain better understanding of dehydration response in plants, we have developed a differential phosphoproteome in a food legume, chickpea (Cicer arietinum L.). Three-week-old chickpea seedlings were subjected to progressive dehydration by withdrawing water, and the changes in the phosphorylation status of a large repertoire of proteins were monitored. The proteins were resolved by 2-DE and stained with phosphospecific fluorescent Pro-Q Diamond dye. Mass spectrometric analysis led to the identification of 91 putative phosphoproteins, presumably involved in a variety of functions including cell defense and rescue, photosynthesis and photorespiration, molecular chaperones, and ion transport, among others. Multiple sites of phosphorylation were predicted on several key elements, which include both the regulatory as well as the functional proteins. A critical survey of the phosphorylome revealed a DREPP (developmentally regulated plasma membrane protein) plasma membrane polypeptide family protein, henceforth designated CaDREPP1. The transcripts of CaDREPP1 were found to be differentially regulated under dehydration stress, further corroborating the proteomic results. This work provides new insights into the possible phosphorylation events triggered by the conditions of progressive water-deficit in plants.

  12. Site-Specific Ser/Thr/Tyr Phosphoproteome of Sinorhizobium meliloti at Stationary Phase.

    PubMed

    Liu, Tao; Tian, Chang Fu; Chen, Wen Xin

    2015-01-01

    Sinorhizobium meliloti, a facultative microsymbiont of alfalfa, should fine-tune its cellular processes to live saprophytically in soils characterized with limited nutrients and diverse stresses. In this study, TiO2 enrichment and LC-MS/MS were used to uncover the site-specific Ser/Thr/Tyr phosphoproteome of S. meliloti in minimum medium at stationary phase. There are a total of 96 unique phosphorylated sites, with a Ser/Thr/Tyr distribution of 63:28:5, in 77 proteins. Phosphoproteins identified in S. meliloti showed a wide distribution pattern regarding to functional categories, such as replication, transcription, translation, posttranslational modification, transport and metabolism of amino acids, carbohydrate, inorganic ion, succinoglycan etc. Ser/Thr/Tyr phosphosites identified within the conserved motif in proteins of key cellular function indicate a crucial role of phosphorylation in modulating cellular physiology. Moreover, phosphorylation in proteins involved in processes related to rhizobial adaptation was also discussed, such as those identified in SMa0114 and PhaP2 (polyhydroxybutyrate synthesis), ActR (pH stress and microaerobic adaption), SupA (potassium stress), chaperonin GroEL2 (viability and potentially symbiosis), and ExoP (succinoglycan synthesis and secretion). These Ser/Thr/Tyr phosphosites identified herein would be helpful for our further investigation and understanding of the role of phosphorylation in rhizobial physiology.

  13. Deoxygenation affects tyrosine phosphoproteome of red cell membrane from patients with sickle cell disease.

    PubMed

    Siciliano, Angela; Turrini, Franco; Bertoldi, Mariarita; Matte, Alessandro; Pantaleo, Antonella; Olivieri, Oliviero; De Franceschi, Lucia

    2010-04-15

    Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder related to the production of a defective form of hemoglobin, hemoglobin S (HbS). One of the hallmarks of SCD is the presence of dense, dehydrate highly adhesive sickle red blood cells (RBCs) that result from persistent membrane damage associated with HbS polymerization, abnormal activation of membrane cation transports and generation of distorted and rigid red cells with membrane perturbation and cytoskeleton dysfunction. Although modulation of phosphorylation state of the proteins from membrane and cytoskeleton networks has been proposed to participate in red cell homeostasis, much still remains to be investigated in normal and diseased red cells. Here, we report that tyrosine (Tyr-) phosphoproteome of sickle red cells was different from normal controls and was affected by deoxygenation. We found proteins, p55 and band 4.1, from the junctional complex, differently Tyr-phosphorylated in SCD RBCs compared to normal RBCs under normoxia and modulated by deoxygenation, while band 4.2 was similarly Tyr-phosphorylated in both conditions. In SCD RBCs we identified the phosphopeptides for protein 4.1R located in the protein FERM domain (Tyr-13) and for alpha-spectrin located near or in a linker region (Tyr-422 and Tyr-1498) involving protein areas crucial for their functions in the context of red cell membrane properties, suggesting that Tyr-phosphorylation may be part of the events involved in maintaining membrane mechanical stability in SCD red cells.

  14. Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases

    PubMed Central

    Steger, Martin; Tonelli, Francesca; Ito, Genta; Davies, Paul; Trost, Matthias; Vetter, Melanie; Wachter, Stefanie; Lorentzen, Esben; Duddy, Graham; Wilson, Stephen; Baptista, Marco AS; Fiske, Brian K; Fell, Matthew J; Morrow, John A; Reith, Alastair D; Alessi, Dario R; Mann, Matthias

    2016-01-01

    Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD. DOI: http://dx.doi.org/10.7554/eLife.12813.001 PMID:26824392

  15. Newly fabricated magnetic lanthanide oxides core-shell nanoparticles in phosphoproteomics.

    PubMed

    Jabeen, Fahmida; Najam-Ul-Haq, Muhammad; Rainer, Matthias; Güzel, Yüksel; Huck, Christian W; Bonn, Guenther K

    2015-01-01

    Metal oxides show high selectivity and sensitivity toward mass spectrometry based enrichment strategies. Phosphopeptides/phosphoproteins enrichment from biological samples is cumbersome because of their low abundance. Phosphopeptides are of interest in enzymes and phosphorylation pathways which lead to the clinical links of a disease. Magnetic core-shell lanthanide oxide nanoparticles (Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3) are fabricated, characterized by SEM, FTIR, and EDX and employed in the enrichment of phosphopeptides. The nanoparticles enrich phosphopeptides from casein variants, nonfat milk, egg yolk, human serum and HeLa cell extract. The materials and enrichment protocols are designed in a way that there are almost no nonspecific bindings. The selectivity is achieved up to 1:8500 using β-casein/BSA mixture and sensitivity down to 1 atto-mole. Batch-to-batch reproducibility is high with the reuse of core-shell nanoparticles up to four cycles. The enrichment followed by MALDI-MS analyses is carried out for the identification of phosphopeptides from serum digest and HeLa cell extract. Characteristic phosphopeptides of phosphoproteins are identified from human serum after the enrichment, which have the diagnostic potential toward prostate cancer. Thus, the lanthanide based magnetic core-shell materials offer a highly selective and sensitive workflow in phosphoproteomics.

  16. The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

    DOE PAGES

    Xiong, Yi; Coradetti, Samuel T.; Li, Xin; ...

    2014-05-29

    Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ) -based LC-MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggestsmore » that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium versus sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Finally, we found mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.« less

  17. The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

    SciTech Connect

    Xiong, Yi; Coradetti, Samuel T.; Li, Xin; Gritsenko, Marina A.; Clauss, Therese; Petyuk, Vlad; Camp, David; Smith, Richard; Cate, Jamie H. D.; Yang, Feng; Glass, N. Louise

    2014-05-29

    Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ) -based LC-MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggests that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium versus sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Finally, we found mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.

  18. Phosphoproteome and Transcriptome of RA-Responsive and RA-Resistant Breast Cancer Cell Lines

    PubMed Central

    Carrier, Marilyn; Joint, Mathilde; Lutzing, Régis; Page, Adeline; Rochette-Egly, Cécile

    2016-01-01

    Retinoic acid (RA), the main active vitamin A metabolite, controls multiple biological processes such as cell proliferation and differentiation through genomic programs and kinase cascades activation. Due to these properties, RA has proven anti-cancer capacity. Several breast cancer cells respond to the antiproliferative effects of RA, while others are RA-resistant. However, the overall signaling and transcriptional pathways that are altered in such cells have not been elucidated. Here, in a large-scale analysis of the phosphoproteins and in a genome-wide analysis of the RA-regulated genes, we compared two human breast cancer cell lines, a RA-responsive one, the MCF7 cell line, and a RA-resistant one, the BT474 cell line, which depicts several alterations of the “kinome”. Using high-resolution nano-LC-LTQ-Orbitrap mass spectrometry associated to phosphopeptide enrichment, we found that several proteins involved in signaling and in transcription, are differentially phosphorylated before and after RA addition. The paradigm of these proteins is the RA receptor α (RARα), which was phosphorylated in MCF7 cells but not in BT474 cells after RA addition. The panel of the RA-regulated genes was also different. Overall our results indicate that RA resistance might correlate with the deregulation of the phosphoproteome with consequences on gene expression. PMID:27362937

  19. Renal artery dissection following marathon running.

    PubMed

    Iqbal, Fahad M; Goparaju, Madhavi; Yemme, Soumya; Lewis, Bruce E

    2009-01-01

    A 38-year-old, previously healthy man presented with flank pain after competing in a marathon. Initial laboratory tests and urinalysis were essentially normal. Both contrast enhanced-computed tomography and magnetic resonance angiography showed an infarcted region of the left lower kidney without renal artery dissection. Thromboembolism was suspected, but further testing was negative. The diagnosis of renal artery dissection was established by angiogram, showing dissection of the segmental branch. The patient remained normotensive, maintained normal renal function, and had resolution of pain symptoms prior to discharge. On the basis of our experience and review of the literature, renal artery dissection occurs in otherwise healthy men and often goes undiagnosed. The management strategy tends to be conservative unless the patient develops progressive decline in renal function or worsening hypertension, with an excellent prognosis. This case also shows the importance of discussing the pros and cons of extreme physical exertion with all patients.

  20. Spontaneous Isolated Superior Mesenteric Artery Dissection

    PubMed Central

    Nath, Anand; Yewale, Sayali; Kousha, Mohammad

    2016-01-01

    A true isolated superior mesenteric artery (SMA) dissection is a rare occurrence. The increasing use of diagnostic imaging studies has resulted in this rare disease being more recognized. A 68-year-old Caucasian female presented with sharp upper abdominal pain. Computed tomography (CT) of the abdomen showed dissection with thrombosis in the proximal SMA. Conservative management with bowel rest, blood pressure control, and anticoagulation relieved her symptoms. Follow-up CT showed stable dissection. Physicians should consider the diagnosis of isolated spontaneous SMA dissection after excluding more common causes. The optimal management pathway has not been firmly established. Conservative management with anticoagulation appears to be a safe first-line therapy in selected patients. PMID:28203123

  1. Animal Rights Groups Target High School Dissection.

    ERIC Educational Resources Information Center

    Trotter, Andrew

    1992-01-01

    Two groups leading the charge against dissection are People for the Ethical Treatment of Animals (PETA) and the Student Action Corps for Animals (SACA). Protests by student and community members remain the movement's strongest weapon. (MLF)

  2. Neck dissection: current concepts and future directions.

    PubMed

    Rigual, Nestor R; Wiseman, Sam M

    2004-01-01

    For individuals diagnosed with head and neck cancer, neck dissection may be performed for therapy or disease staging. The classification of neck dissection and the definition of precise anatomic landmarks have allowed for this operation, and its many variations, to become standardized world-wide. SLNBX shows promise in its ability to accurately stage NO head and neck cancer and may allow patients with no micro metastatic disease to avoid neck dissection. Before this technique becomes adopted into routine clinical practice, however, it must first be prospectively scrutinized in large patient populations. Regardless of the future role of SLNBX in the management of head and neck cancer, currently it is only through a complete understanding of the clinical, theoretic, and technical aspects of neck dis-section that surgeons may benefit individual patients and the head and neck cancer patient population as a whole.

  3. A New Idea for Dissecting Tray

    ERIC Educational Resources Information Center

    Branham, Arthur

    1976-01-01

    A method of preparing a special dissecting tray to be used with transmitted light as well as reflected light is presented. It may also be used with an overhead projector to illustrate some skeletal structures in vertebrates. (Author/EB)

  4. [Gross anatomy dissection and the legal control].

    PubMed

    Yamashina, Shohei; Shibata, Yosaburo

    2010-12-01

    In Japan, dissection of human body is generally prohibited by the Penal Code, i.e. the criminal law. However, the Postmortem Examination and Corpse Preservation Act allows for the dissection of the body in very limited situations, that include gross anatomy dissection and pathological and forensic autopsy in medical and dental schools. Growing numbers of co-medical schools have been founded more recently in Japan, and not a small number of co-medical schools try to adopt human body dissection in the course of anatomy education. The present short communication reminds us of the ways of thinking of the Postmortem Examination and Corpse Preservation Act and the Act on Body Donation for Medical and Dental Education in order that anatomy education in medical as well as co-medical schools takes place under the regulation by these two laws.

  5. Managing Dissections of the Thoracic Aorta

    PubMed Central

    WONG, DANIEL R.; LEMAIRE, SCOTT A.; COSELLI, JOSEPH S.

    2010-01-01

    Thoracic aortic dissection is associated with substantial morbidity and mortality, and it requires timely and accurate diagnosis and treatment. Long-term antihypertensive therapy remains critical for the treatment of this disease. Surgical intervention, although still a formidable undertaking, has evolved to better address both acute and chronic dissection, and the results have improved. Basic and clinical research, as well as technological advances, have increased our understanding of this challenging disease state. PMID:18481490

  6. Recurrent post-partum coronary artery dissection

    PubMed Central

    2010-01-01

    Coronary artery dissection is a rare but well-described cause for myocardial infarction during the post-partum period. Dissection of multiple coronary arteries is even less frequent. Here we present a case of recurrent post-partum coronary artery dissections. This unusual presentation poses unique problems for management. A 35 year-old female, gravida 3 para 2, presented with myocardial infarction 9 weeks and 3 days post-partum. Cardiac catheterization demonstrated left anterior descending (LAD) dissection but an otherwise normal coronary anatomy. The lesion was treated with four everolimus eluting stents. Initially the patient made an unremarkable recovery until ventricular fibrillation arrest occurred on the following day. Unsynchronized cardioversion restored a normal sinus rhythm and repeat catheterization revealed new right coronary artery (RCA) dissection. A wire was passed distally, but it was unclear whether this was through the true or false lumen and no stents could be placed. However, improvement of distal RCA perfusion was noted on angiogram. Despite failure of interventional therapy the patient was therefore treated conservatively. Early operation after myocardial infarction has a significantly elevated risk of mortality and the initial dissection had occurred within 24 hours. This strategy proved successful as follow-up transthoracic echocardiography after four months demonstrated a preserved left ventricular ejection fraction of 55-60% without regional wall motion abnormalities. The patient remained asymptomatic from a cardiac point of view. PMID:20932332

  7. Recurrent post-partum coronary artery dissection.

    PubMed

    Rajab, Taufiek K; Khalpey, Zain; Kraemer, Bernhard; Resnic, Frederic S; Gallegos, Robert P

    2010-10-09

    Coronary artery dissection is a rare but well-described cause for myocardial infarction during the post-partum period. Dissection of multiple coronary arteries is even less frequent. Here we present a case of recurrent post-partum coronary artery dissections. This unusual presentation poses unique problems for management. A 35 year-old female, gravida 3 para 2, presented with myocardial infarction 9 weeks and 3 days post-partum. Cardiac catheterization demonstrated left anterior descending (LAD) dissection but an otherwise normal coronary anatomy. The lesion was treated with four everolimus eluting stents. Initially the patient made an unremarkable recovery until ventricular fibrillation arrest occurred on the following day. Unsynchronized cardioversion restored a normal sinus rhythm and repeat catheterization revealed new right coronary artery (RCA) dissection. A wire was passed distally, but it was unclear whether this was through the true or false lumen and no stents could be placed. However, improvement of distal RCA perfusion was noted on angiogram. Despite failure of interventional therapy the patient was therefore treated conservatively. Early operation after myocardial infarction has a significantly elevated risk of mortality and the initial dissection had occurred within 24 hours. This strategy proved successful as follow-up transthoracic echocardiography after four months demonstrated a preserved left ventricular ejection fraction of 55-60% without regional wall motion abnormalities. The patient remained asymptomatic from a cardiac point of view.

  8. iPhos: a toolkit to streamline the alkaline phosphatase-assisted comprehensive LC-MS phosphoproteome investigation

    PubMed Central

    2014-01-01

    Background Comprehensive characterization of the phosphoproteome in living cells is critical in signal transduction research. But the low abundance of phosphopeptides among the total proteome in cells remains an obstacle in mass spectrometry-based proteomic analysis. To provide a solution, an alternative analytic strategy to confidently identify phosphorylated peptides by using the alkaline phosphatase (AP) treatment combined with high-resolution mass spectrometry was provided. While the process is applicable, the key integration along the pipeline was mostly done by tedious manual work. Results We developed a software toolkit, iPhos, to facilitate and streamline the work-flow of AP-assisted phosphoproteome characterization. The iPhos tookit includes one assister and three modules. The iPhos Peak Extraction Assister automates the batch mode peak extraction for multiple liquid chromatography mass spectrometry (LC-MS) runs. iPhos Module-1 can process the peak lists extracted from the LC-MS analyses derived from the original and dephosphorylated samples to mine out potential phosphorylated peptide signals based on mass shift caused by the loss of some multiples of phosphate groups. And iPhos Module-2 provides customized inclusion lists with peak retention time windows for subsequent targeted LC-MS/MS experiments. Finally, iPhos Module-3 facilitates to link the peptide identifications from protein search engines to the quantification results from pattern-based label-free quantification tools. We further demonstrated the utility of the iPhos toolkit on the data of human metastatic lung cancer cells (CL1-5). Conclusions In the comparison study of the control group of CL1-5 cell lysates and the treatment group of dasatinib-treated CL1-5 cell lysates, we demonstrated the applicability of the iPhos toolkit and reported the experimental results based on the iPhos-facilitated phosphoproteome investigation. And further, we also compared the strategy with pure DDA-based LC

  9. Quantitative phosphoproteomic analysis identifies activation of the RET and IGF-1R/IR signaling pathways in neuroblastoma.

    PubMed

    DeNardo, Bradley D; Holloway, Michael P; Ji, Qinqin; Nguyen, Kevin T; Cheng, Yan; Valentine, Marcus B; Salomon, Arthur; Altura, Rachel A

    2013-01-01

    Neuroblastoma is an embryonal tumor of childhood with a heterogenous clinical presentation that reflects differences in activation of complex biological signaling pathways. Protein phosphorylation is a key component of cellular signal transduction and plays a critical role in processes that control cancer cell growth and survival. We used shotgun LC/MS to compare phosphorylation between a human MYCN amplified neuroblastoma cell line (NB10), modeling a resistant tumor, and a human neural precursor cell line (NPC), modeling a normal baseline neural crest cell. 2181 unique phosphorylation sites representing 1171 proteins and 2598 phosphopeptides were found. Protein kinases accounted for 6% of the proteome, with a predominance of tyrosine kinases, supporting their prominent role in oncogenic signaling pathways. Highly abundant receptor tyrosine kinase (RTK) phosphopeptides in the NB10 cell line relative to the NPC cell line included RET, insulin-like growth factor 1 receptor/insulin receptor (IGF-1R/IR), and fibroblast growth factor receptor 1 (FGFR1). Multiple phosphorylated peptides from downstream mediators of the PI3K/AKT/mTOR and RAS pathways were also highly abundant in NB10 relative to NPC. Our analysis highlights the importance of RET, IGF-1R/IR and FGFR1 as RTKs in neuroblastoma and suggests a methodology that can be used to identify potential novel biological therapeutic targets. Furthermore, application of this previously unexploited technology in the clinic opens the possibility of providing a new wide-scale molecular signature to assess disease progression and prognosis.

  10. The Effect of Animal Dissections on Student Acquisition of Knowledge of and Attitudes toward the Animals Dissected.

    ERIC Educational Resources Information Center

    McCollum, Terry L.

    A conflict exists over the use of animals in the classroom. One aspect of this use involved the dissection of animals. Animal protection advocates report that dissections constitute abuse of the animals dissected. The advocates state that what is learned by dissection could be more effectively learned by other means. Some science educators state…

  11. Evaluating dissection in the gross anatomy course: Correlation between quality of laboratory dissection and students outcomes.

    PubMed

    Nwachukwu, Chika; Lachman, Nirusha; Pawlina, Wojciech

    2015-01-01

    Anatomy learned by active exploration through dissection has many proven benefits including improvement of anatomic knowledge. Decreased laboratory time may affect the quality of dissection and ultimately lower student performance in anatomy translating to lower knowledge acquisition. The aim of this study was to determine whether the quality of students' dissection in teams correlates with their performance in the gross anatomy course. Quality of dissections for each team enrolled in a gross anatomy course at Mayo Medical School was evaluated biweekly using a five-point rubric based on course learning objectives. Assessment of anatomic knowledge was based on sequential laboratory practice practical examination scores, achievements on daily audience response system (ARS) quizzes, and final practical, written, and National Board of Medical Examiners(®) (NBME(®) ) Gross Anatomy and Embryology Subject Examinations. Twelve teams comprising 48 students were included in the study. There was a positive correlation between dissection quality and practice practical examination score (R = 0.83) and a negative correlation between dissection quality and ARS quizzes (R = -0.985). Dissection teams with a passing score on their dissection evaluations (>70%) performed better on their final examinations. Based on an end of course survey, students agreed that dissection evaluations should continue to be a part of the course. This study showed that better quality of dissection was associated with higher scores on practice practical examinations, final practical, written, and NBME examinations. The study demonstrated a positive correlation between dissection evaluations, accompanied by formative feedback during the course, and higher scores on final course assessments.

  12. Dissection and dissection-associated required experiences improve student performance in gross anatomy: Differences among quartiles.

    PubMed

    Pizzimenti, Marc A; Pantazis, Nicholas; Sandra, Alexander; Hoffmann, Darren S; Lenoch, Susan; Ferguson, Kristi J

    2016-05-06

    To promote student learning, educational strategies should provide multiple levels of engagement with the subject matter. This study investigated examination data from five first year medical gross anatomy class cohorts (692 students) to determine if enhanced student performance was correlated with learning through dissection in a course that used a rotating dissection schedule coupled with peer teaching and other associated experiences. When students performed two of five weekly dissections for a given unit, their average scores on both laboratory and written examinations tended to increase as compared to when they had completed only one week of dissection (P < 0.01). However, these performance gains differed across the class strata and were related to the amount of dissection completed. Students in the upper quartile (UQS) of the class benefited when they had dissected once (92.8%) or twice (92.4%), and these scores were significantly higher than those attained when learning from peers (90.3%, P < 0.01). Students in the lower quartile (LQS) benefited most from the dissection experiences, where practical examination performance was better (77.8% and 80.5%) than when these students learned material from their peers (73.7%, P < 0.01). Although UQS benefited from dissection, LQS benefited to a greater extent in both the practical and written examinations with dissection. Although limited, these data suggest that dissection, coupled with associated educational activities, is an effective pedagogical strategy for learning. Further investigation is required to evaluate the concomitant benefits of peer teaching that are associated with the dissection experience. Anat Sci Educ 9: 238-246. © 2015 American Association of Anatomists.

  13. Predicting flow in aortic dissection: comparison of computational model with PC-MRI velocity measurements.

    PubMed

    Cheng, Z; Juli, C; Wood, N B; Gibbs, R G J; Xu, X Y

    2014-09-01

    Aortic dissection is a life-threatening process in which the weakened wall develops a tear, causing separation of wall layers. The dissected layers separate the original true aortic lumen and a newly created false lumen. If untreated, the condition can be fatal. Flow rate in the false lumen is a key feature for false lumen patency, which has been regarded as one of the most important predictors of adverse early and later outcomes. Detailed flow analysis in the dissected aorta may assist vascular surgeons in making treatment decisions, but computational models to simulate flow in aortic dissections often involve several assumptions. The purpose of this study is to assess the computational models adopted in previous studies by comparison with in vivo velocity data obtained by means of phase-contrast magnetic resonance imaging (PC-MRI). Aortic dissection geometry was reconstructed from computed tomography (CT) images, while PC-MRI velocity data were used to define inflow conditions and to provide distal velocity components for comparison with the simulation results. The computational fluid dynamics (CFD) simulation incorporated a laminar-turbulent transition model, which is necessary for adequate flow simulation in aortic conditions. Velocity contours from PC-MRI and CFD in the two lumens at the distal plane were compared at four representative time points in the pulse cycle. The computational model successfully captured the complex regions of flow reversal and recirculation qualitatively, although quantitative differences exist. With a rigid wall assumption and exclusion of arch branches, the CFD model over-predicted the false lumen flow rate by 25% at peak systole. Nevertheless, an overall good agreement was achieved, confirming the physiological relevance and validity of the computational model for type B aortic dissection with a relatively stiff dissection flap.

  14. Science Teachers and the Dissection Debate: Perspectives on Animal Dissection and Alternatives

    ERIC Educational Resources Information Center

    Oakley, Jan

    2012-01-01

    This study investigated Ontario science and biology teachers' practices and attitudes toward animal dissection and dissection alternatives. The data was collected through a mixed methods approach involving online surveys (n = 153) and subsequent telephone interviews (n = 9) with secondary school science and biology teachers. The findings indicate…

  15. Salt-induced changes in cardiac phosphoproteome in a rat model of chronic renal failure.

    PubMed

    Su, Zhengxiu; Zhu, Hongguo; Zhang, Menghuan; Wang, Liangliang; He, Hanchang; Jiang, Shaoling; Hou, Fan Fan; Li, Aiqing

    2014-01-01

    Heart damage is widely present in patients with chronic kidney disease. Salt diet is the most important environmental factor affecting development of chronic renal failure and cardiovascular diseases. The proteins involved in chronic kidney disease -induced heart damage, especially their posttranslational modifications, remain largely unknown to date. Sprague-Dawley rats underwent 5/6 nephrectomy (chronic renal failure model) or sham operation were treated for 2 weeks with a normal-(0.4% NaCl), or high-salt (4% NaCl) diet. We employed TiO2 enrichment, iTRAQ labeling and liquid-chromatography tandem mass spectrometry strategy for phosphoproteomic profiling of left ventricular free walls in these animals. A total of 1724 unique phosphopeptides representing 2551 non-redundant phosphorylation sites corresponding to 763 phosphoproteins were identified. During normal salt feeding, 89 (54%) phosphopeptides upregulated and 76 (46%) phosphopeptides downregulated in chronic renal failure rats relative to sham rats. In chronic renal failure rats, high salt intake induced upregulation of 84 (49%) phosphopeptides and downregulation of 88 (51%) phosphopeptides. Database searches revealed that most of the identified phospholproteins were important signaling molecules such as protein kinases, receptors and phosphatases. These phospholproteins were involved in energy metabolism, cell communication, cell differentiation, cell death and other biological processes. The Search Tool for the Retrieval of Interacting Genes analysis revealed functional links among 15 significantly regulated phosphoproteins in chronic renal failure rats compared to sham group, and 23 altered phosphoproteins induced by high salt intake. The altered phosphorylation levels of two proteins involved in heart damage, lamin A and phospholamban were validated. Expression of the downstream genes of these two proteins, desmin and SERCA2a, were also analyzed.

  16. Phosphoproteomic analysis of the non-seed vascular plant model Selaginella moellendorffii

    PubMed Central

    2014-01-01

    Background Selaginella (Selaginella moellendorffii) is a lycophyte which diverged from other vascular plants approximately 410 million years ago. As the first reported non-seed vascular plant genome, Selaginella genome data allow comparative analysis of genetic changes that may be associated with land plant evolution. Proteomics investigations on this lycophyte model have not been extensively reported. Phosphorylation represents the most common post-translational modifications and it is a ubiquitous regulatory mechanism controlling the functional expression of proteins inside living organisms. Results In this study, polyethylene glycol fractionation and immobilized metal ion affinity chromatography were employed to isolate phosphopeptides from wild-growing Selaginella. Using liquid chromatography-tandem mass spectrometry analysis, 1593 unique phosphopeptides spanning 1104 non-redundant phosphosites with confirmed localization on 716 phosphoproteins were identified. Analysis of the Selaginella dataset revealed features that are consistent with other plant phosphoproteomes, such as the relative proportions of phosphorylated Ser, Thr, and Tyr residues, the highest occurrence of phosphosites in the C-terminal regions of proteins, and the localization of phosphorylation events outside protein domains. In addition, a total of 97 highly conserved phosphosites in evolutionary conserved proteins were identified, indicating the conservation of phosphorylation-dependent regulatory mechanisms in phylogenetically distinct plant species. On the other hand, close examination of proteins involved in photosynthesis revealed phosphorylation events which may be unique to Selaginella evolution. Furthermore, phosphorylation motif analyses identified Pro-directed, acidic, and basic signatures which are recognized by typical protein kinases in plants. A group of Selaginella-specific phosphoproteins were found to be enriched in the Pro-directed motif class. Conclusions Our work provides

  17. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection

    SciTech Connect

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.; Petyuk, Vladislav A.; Jones, Marcus B.; Gritsenko, Marina A.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2013-09-22

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared to the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin (TTP), a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of TTP, leading to the production of cytokines such as IL-1beta and TNF-alpha which may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that controls infection by this pathogen.

  18. Phosphoproteomics reveals the effect of ethylene in soybean root under flooding stress.

    PubMed

    Yin, Xiaojian; Sakata, Katsumi; Komatsu, Setsuko

    2014-12-05

    Flooding has severe negative effects on soybean growth. To explore the flooding-responsive mechanisms in early-stage soybean, a phosphoproteomic approach was used. Two-day-old soybean plants were treated without or with flooding for 3, 6, 12, and 24 h, and root tip proteins were then extracted and analyzed at each time point. After 3 h of flooding exposure, the fresh weight of soybeans increased, whereas the ATP content of soybean root tips decreased. Using a gel-free proteomic technique, a total of 114 phosphoproteins were identified in the root tip samples, and 34 of the phosphoproteins were significantly changed with respect to phosphorylation status after 3 h of flooding stress. Among these phosphoproteins, eukaryotic translation initiation factors were dephosphorylated, whereas several protein synthesis-related proteins were phosphorylated. The mRNA expression levels of sucrose phosphate synthase 1F and eukaryotic translation initiation factor 4 G were down-regulated, whereas UDP-glucose 6-dehydrogenase mRNA expression was up-regulated during growth but down-regulated under flooding stress. Furthermore, bioinformatic protein interaction analysis of flooding-responsive proteins based on temporal phosphorylation patterns indicated that eukaryotic translation initiation factor 4 G was located in the center of the network during flooding. Soybean eukaryotic translation initiation factor 4 G has homology to programmed cell death 4 protein and is implicated in ethylene signaling. The weight of soybeans was increased with treatment by an ethylene-releasing agent under flooding condition, but it was decreased when plants were exposed to an ethylene receptor antagonist. These results suggest that the ethylene signaling pathway plays an important role, via the protein phosphorylation, in mechanisms of plant tolerance to the initial stages of flooding stress in soybean root tips.

  19. Identifying drug effects via pathway alterations using an integer linear programming optimization formulation on phosphoproteomic data.

    PubMed

    Mitsos, Alexander; Melas, Ioannis N; Siminelakis, Paraskeuas; Chairakaki, Aikaterini D; Saez-Rodriguez, Julio; Alexopoulos, Leonidas G

    2009-12-01

    Understanding the mechanisms of cell function and drug action is a major endeavor in the pharmaceutical industry. Drug effects are governed by the intrinsic properties of the drug (i.e., selectivity and potency) and the specific signaling transduction network of the host (i.e., normal vs. diseased cells). Here, we describe an unbiased, phosphoproteomic-based approach to identify drug effects by monitoring drug-induced topology alterations. With our proposed method, drug effects are investigated under diverse stimulations of the signaling network. Starting with a generic pathway made of logical gates, we build a cell-type specific map by constraining it to fit 13 key phopshoprotein signals under 55 experimental conditions. Fitting is performed via an Integer Linear Program (ILP) formulation and solution by standard ILP solvers; a procedure that drastically outperforms previous fitting schemes. Then, knowing the cell's topology, we monitor the same key phosphoprotein signals under the presence of drug and we re-optimize the specific map to reveal drug-induced topology alterations. To prove our case, we make a topology for the hepatocytic cell-line HepG2 and we evaluate the effects of 4 drugs: 3 selective inhibitors for the Epidermal Growth Factor Receptor (EGFR) and a non-selective drug. We confirm effects easily predictable from the drugs' main target (i.e., EGFR inhibitors blocks the EGFR pathway) but we also uncover unanticipated effects due to either drug promiscuity or the cell's specific topology. An interesting finding is that the selective EGFR inhibitor Gefitinib inhibits signaling downstream the Interleukin-1alpha (IL1alpha) pathway; an effect that cannot be extracted from binding affinity-based approaches. Our method represents an unbiased approach to identify drug effects on small to medium size pathways which is scalable to larger topologies with any type of signaling interventions (small molecules, RNAi, etc). The method can reveal drug effects on

  20. Chronic low-dose-rate ionising radiation affects the hippocampal phosphoproteome in the ApoE−/− Alzheimer's mouse model

    PubMed Central

    Kempf, Stefan J.; Janik, Dirk; Barjaktarovic, Zarko; Braga-Tanaka, Ignacia; Tanaka, Satoshi; Neff, Frauke; Saran, Anna; Larsen, Martin R.; Tapio, Soile

    2016-01-01

    Accruing data indicate that radiation-induced consequences resemble pathologies of neurodegenerative diseases such as Alzheimer's. The aim of this study was to elucidate the effect on hippocampus of chronic low-dose-rate radiation exposure (1 mGy/day or 20 mGy/day) given over 300 days with cumulative doses of 0.3 Gy and 6.0 Gy, respectively. ApoE deficient mutant C57Bl/6 mouse was used as an Alzheimer's model. Using mass spectrometry, a marked alteration in the phosphoproteome was found at both dose rates. The radiation-induced changes in the phosphoproteome were associated with the control of synaptic plasticity, calcium-dependent signalling and brain metabolism. An inhibition of CREB signalling was found at both dose rates whereas Rac1-Cofilin signalling was found activated only at the lower dose rate. Similarly, the reduction in the number of activated microglia in the molecular layer of hippocampus that paralleled with reduced levels of TNFα expression and lipid peroxidation was significant only at the lower dose rate. Adult neurogenesis, investigated by Ki67, GFAP and NeuN staining, and cell death (activated caspase-3) were not influenced at any dose or dose rate. This study shows that several molecular targets induced by chronic low-dose-rate radiation overlap with those of Alzheimer's pathology. It may suggest that ionising radiation functions as a contributing risk factor to this neurodegenerative disease. PMID:27708245

  1. Meta-Analysis of Arabidopsis thaliana Phospho-Proteomics Data Reveals Compartmentalization of Phosphorylation Motifs[C][W

    PubMed Central

    van Wijk, Klaas J.; Friso, Giulia; Walther, Dirk; Schulze, Waltraud X.

    2014-01-01

    Protein (de)phosphorylation plays an important role in plants. To provide a robust foundation for subcellular phosphorylation signaling network analysis and kinase-substrate relationships, we performed a meta-analysis of 27 published and unpublished in-house mass spectrometry–based phospho-proteome data sets for Arabidopsis thaliana covering a range of processes, (non)photosynthetic tissue types, and cell cultures. This resulted in an assembly of 60,366 phospho-peptides matching to 8141 nonredundant proteins. Filtering the data for quality and consistency generated a set of medium and a set of high confidence phospho-proteins and their assigned phospho-sites. The relation between single and multiphosphorylated peptides is discussed. The distribution of p-proteins across cellular functions and subcellular compartments was determined and showed overrepresentation of protein kinases. Extensive differences in frequency of pY were found between individual studies due to proteomics and mass spectrometry workflows. Interestingly, pY was underrepresented in peroxisomes but overrepresented in mitochondria. Using motif-finding algorithms motif-x and MMFPh at high stringency, we identified compartmentalization of phosphorylation motifs likely reflecting localized kinase activity. The filtering of the data assembly improved signal/noise ratio for such motifs. Identified motifs were linked to kinases through (bioinformatic) enrichment analysis. This study also provides insight into the challenges/pitfalls of using large-scale phospho-proteomic data sets to nonexperts. PMID:24894044

  2. Biosynthesis and Regulation of Wheat Amylose and Amylopectin from Proteomic and Phosphoproteomic Characterization of Granule-binding Proteins

    PubMed Central

    Chen, Guan-Xing; Zhou, Jian-Wen; Liu, Yan-Lin; Lu, Xiao-Bing; Han, Cai-Xia; Zhang, Wen-Ying; Xu, Yan-Hao; Yan, Yue-Ming

    2016-01-01

    Waxy starch has an important influence on the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and grain yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Our results revealed the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis. PMID:27604546

  3. In vivo Phosphoproteome of Human Skeletal Muscle Revealed by Phosphopeptide Enrichment and HPLC-ESI-MS/MS

    PubMed Central

    Højlund, Kurt; Bowen, Benjamin P.; Hwang, Hyonson; Flynn, Charles R.; Madireddy, Lohith; Thangiah, Geetha; Langlais, Paul; Meyer, Christian; Mandarino, Lawrence J.; Yi, Zhengping

    2009-01-01

    Protein phosphorylation plays an essential role in signal transduction pathways that regulate substrate and energy metabolism, contractile function, and muscle mass in human skeletal muscle. Abnormal phosphorylation of signaling enzymes has been identified in insulin resistant muscle using phosphoepitope-specific antibodies, but its role in other skeletal muscle disorders remains largely unknown. This may be in part due to insufficient knowledge of relevant targets. Here, we therefore present the first large-scale in vivo phosphoproteomic study of human skeletal muscle from 3 lean, healthy volunteers. Trypsin digestion of 3-5 mg human skeletal muscle protein was followed by phosphopeptide enrichment using SCX and TiO2. The resulting phosphopeptides were analyzed by HPLC-ESI-MS/MS. Using this unbiased approach, we identified 306 distinct in vivo phosphorylation sites in 127 proteins, including 240 phosphoserines, 53 phosphothreonines and 13 phosphotyrosines in at least 2 out of 3 subjects. In addition, 61 ambiguous phosphorylation sites were identified in at least 2 out of 3 subjects. The majority of phosphoproteins detected are involved in sarcomeric function, excitation-contraction coupling (the Ca2+-cycle), glycolysis and glycogen metabolism. Of particular interest, we identified multiple novel phosphorylation sites on several sarcomeric Z-disc proteins known to be involved in signaling and muscle disorders. These results provide numerous new targets for the investigation of human skeletal muscle phosphoproteins in health and disease and demonstrate feasibility of phosphoproteomics research of human skeletal muscle in vivo. PMID:19764811

  4. Up-to-Date Workflow for Plant (Phospho)proteomics Identifies Differential Drought-Responsive Phosphorylation Events in Maize Leaves.

    PubMed

    Vu, Lam Dai; Stes, Elisabeth; Van Bel, Michiel; Nelissen, Hilde; Maddelein, Davy; Inzé, Dirk; Coppens, Frederik; Martens, Lennart; Gevaert, Kris; De Smet, Ive

    2016-12-02

    Protein phosphorylation is one of the most common post-translational modifications (PTMs), which can regulate protein activity and localization as well as protein-protein interactions in numerous cellular processes. Phosphopeptide enrichment techniques enable plant researchers to acquire insight into phosphorylation-controlled signaling networks in various plant species. Most phosphoproteome analyses of plant samples still involve stable isotope labeling, peptide fractionation, and demand a lot of mass spectrometry (MS) time. Here, we present a simple workflow to probe, map, and catalogue plant phosphoproteomes, requiring relatively low amounts of starting material, no labeling, no fractionation, and no excessive analysis time. Following optimization of the different experimental steps on Arabidopsis thaliana samples, we transferred our workflow to maize, a major monocot crop, to study signaling upon drought stress. In addition, we included normalization to protein abundance to identify true phosphorylation changes. Overall, we identified a set of new phosphosites in both Arabidopsis thaliana and maize, some of which are differentially phosphorylated upon drought. All data are available via ProteomeXchange with identifier PXD003634, but to provide easy access to our model plant and crop data sets, we created an online database, Plant PTM Viewer ( bioinformatics.psb.ugent.be/webtools/ptm_viewer/ ), where all phosphosites identified in our study can be consulted.

  5. Painless aortic dissection presenting as paraplegia.

    PubMed

    Colak, Necmettin; Nazli, Yunus; Alpay, Mehmet Fatih; Akkaya, Ismail Olgun; Cakir, Omer

    2012-01-01

    Acute dissection of the aorta can be life-threatening. As a presenting manifestation of aortic dissection, neurologic complications such as paraplegia are rare. Herein, we report the case of a 51-year-old man who presented with sudden-onset paraplegia and ischemia of the legs, with no chest or back pain. His medical history included coronary artery bypass grafting. Physical examination revealed pulseless lower extremities, and computed tomography showed aortic dissection from the ascending aorta to the common iliac arteries bilaterally. A lumbar catheter was inserted for cerebrospinal fluid drainage, and axillary arterial cannulation was established. With the use of cardiopulmonary bypass, the aortic dissection was corrected, and the previous coronary artery grafts were reattached. The surgery restored spinal and lower-extremity perfusion, and the patient walked unaided from the hospital upon his discharge 5 days later. Although acute aortic dissection presenting as paraplegia is rare, it should be considered in patients who have pulseless femoral arteries bilaterally and sudden-onset paraplegia, despite no pain in the chest or back. Prompt diagnosis and intervention can prevent morbidity and death.

  6. Management of type A dissection with malperfusion

    PubMed Central

    Yang, Bo; Patel, Himanshu J.; Williams, David M.; Dasika, Narasimham L.

    2016-01-01

    Malperfusion is a common lethal complication of acute aortic dissection following rupture, for which the optimal management strategy has yet to be clearly established. The objective of this study was to reassess the management of acute type A aortic dissection (Type A-AAD) with malperfusion. We retrospectively analyzed the outcomes of all patients with Type A-AAD with malperfusion at the University of Michigan and compared the results from patients that directly underwent open surgical repair versus those who had percutaneous reperfusion prior to open surgical repair. Based on the results, we developed a patient care protocol for the treatment of all patients with acute type A dissection. We later re-analyzed the long-term outcomes for patients using the protocol. The present study demonstrated that, although the outcomes for patients with acute type A aortic dissection with malperfusion syndrome treated with initial percutaneous reperfusion and delayed open surgical intervention are not as good as the results for patients with uncomplicated Type A-AAD that undergo immediate surgical repair, their outcomes continue the long-term outcomes of the former group are superior. To outdo patients with acute type A aortic dissection with malperfusion syndrome treated with immediate open surgical intervention. In conclusion, at the University of Michigan we continue to use our patient care protocol to treat patients with Type A-AAD. PMID:27563540

  7. Dissection of the aorta in Turner's syndrome.

    PubMed Central

    Price, W H; Wilson, J

    1983-01-01

    Three deaths from dissection of the aorta in a series of 157 adult women with Turner's syndrome are reported. These are greatly in excess of the numbers expected. None of the three patients had a coarctation of the aorta. One had aortic regurgitation but there was no reason to believe that the aorta in the other two patients had been subjected to unusual haemodynamic stresses. Cystic medial necrosis of the aorta was described in two patients on whom necropsies were carried out. It is concluded that there is probably a greatly increased risk of dissection of the aorta in Turner's syndrome even in the absence of any other abnormality of the aorta and aortic valve. Previously reported cases of aortic dissection in Turner's syndrome are discussed. PMID:6842536

  8. The nature of dissection: Exploring student conceptions

    NASA Astrophysics Data System (ADS)

    York, Katharine

    The model of conceptual change in science describes the process of learning as a complete restructuring of knowledge, when learners discover or are shown more plausible, intelligent alternatives to existing conceptions. Emotions have been acknowledged as part of a learner's conceptual ecology, but the effects of emotions on learning have yet to be described. This research was conducted to examine the role that emotions have on learning for thirteen high school students, as they dissected cats in a Human Anatomy and Physiology class. The project also investigated whether a student's emotional reactions may be used to develop a sense of connectedness with the nonhuman world, which is defined as ecological literacy. This study utilized a grounded theory approach, in which student responses to interviews were the primary source of data. Interviews were transcribed, and responses were coded according to a constant comparative method of analysis. Responses were compared with the four conditions necessary for conceptual change to occur, and also to five principles of ecological literacy. Students who had negative reactions to dissection participated less in the activity, and demonstrated less conceptual change. Two female students showed the strongest emotional reactions to dissection, and also the lowest amount of conceptual change. One male student also had strong negative reactions to death, and showed no conceptual change. The dissection experiences of the students in this study did not generally reflect ecological principles. The two students whose emotional reactions to dissection were the most negative demonstrated the highest degree of ecological literacy. These results provide empirical evidence of the effects that emotions have on learning, and also supports the opinions of educators who do not favor dissection, because it does not teach students to respect all forms of life.

  9. Intracranial Vertebral Artery Dissections: Evolving Perspectives

    PubMed Central

    Ali, M.S.; Amenta, P.S.; Starke, R.M.; Jabbour, P.M.; Gonzalez, L.F.; Tjoumakaris, S.I.; Flanders, A.E.; Rosenwasser, R.H.; Dumont, A.S.

    2012-01-01

    Summary Intracranial vertebral artery dissection (VAD) represents the underlying etiology in a significant percentage of posterior circulation ischemic strokes and subarachnoid hemorrhages. These lesions are particularly challenging in their diagnosis, management, and in the prediction of long-term outcome. Advances in the understanding of underlying processes leading to dissection, as well as the evolution of modern imaging techniques are discussed. The data pertaining to medical management of intracranial VADs, with emphasis on anticoagulants and antiplatelet agents, is reviewed. Surgical intervention is discussed, including, the selection of operative candidates, open and endovascular procedures, and potential complications. The evolution of endovascular technology and techniques is highlighted. PMID:23217643

  10. Acute thoracic aortic dissection: how to defuse a time bomb.

    PubMed

    McClarren-Curry, C; Shaughnessy, K

    1999-01-01

    Acute thoracic aortic dissection is frequently misdiagnosed, and even with surgical intervention, the mortality rate is 50%. This article focuses on assessment, interventions, and postoperative care of patients with aortic dissection.

  11. Cow's Eye Dissection in the Physics Lab.

    ERIC Educational Resources Information Center

    Lapp, David R.; Keenan, James E.

    1991-01-01

    Proposes the science demonstration of dissecting a cow's eye to integrate biology and physics in the study of optics and lenses. Reviews the anatomy of the eye, describes the visual process and covers topics as index of refraction of the cornea, microscopic receptors, the lens, and the retina. (MDH)

  12. Squid Dissection: From Pen to Ink.

    ERIC Educational Resources Information Center

    Brown, Cindy; Kisiel, Jim

    2003-01-01

    Introduces students to dissection, which is an important part of scientific discovery. Students not only gain an understanding of the anatomy of a squid, but also develop a sense of responsibility and respect for the animal that they are using as a learning tool. (Author/SOE)

  13. Nested Dissection Interface Reconstruction in Pececillo

    SciTech Connect

    Jibben, Zechariah Joel

    2016-08-31

    A nested dissection method for interface reconstruction in a volume tracking framework has been implemented in Pececillo. This method provides a significant improvement over the traditional onion-skin method, which does not appropriately handle T-shaped multimaterial intersections and dynamic contact lines present in additive manufacturing simulations. The resulting implementation lays the groundwork for further re- search in numerical contact angle estimates.

  14. Spontaneous healing of spontaneous coronary artery dissection.

    PubMed

    Almafragi, Amar; Convens, Carl; Heuvel, Paul Van Den

    2010-01-01

    Spontaneous coronary artery dissection (SCAD) is a rare cause of acute coronary syndrome and sudden cardiac death. It should be suspected in every healthy young woman without cardiac risk factors, especially during the peripartum or postpartum periods. It is important to check for a history of drug abuse, collagen vascular disease or blunt trauma of the chest. Coronary angiography is essential for diagnosis and early management. We wonder whether thrombolysis might aggravate coronary dissection. All types of treatment (medical therapy, percutaneous intervention or surgery) improve the prognosis without affecting survival times if used appropriately according to the clinical stability and the angiographic features of the involved coronary arteries. Prompt recognition and targeted treatment improve outcomes. We report a case of SCAD in a young female free of traditional cardiovascular risk factors, who presented six hours after thrombolysis for ST elevation myocardial infarction. Coronary angiography showed a dissection of the left anterior descending and immediate branch. She had successful coronary artery bypass grafting, with complete healing of left anterior descending dissection.

  15. Isolated Posterior Cerebral Artery Dissection: Report of Three Cases

    DTIC Science & Technology

    2005-08-31

    Dissecting intracranial aneurysms usually present with large cerebral infarcts, but may also present with subarachnoid hemorrhage or both (2, 6, 15...diagnosis of intracranial dissection and dissecting aneurysms is predominantly made by DSA, which remains the gold standard technique. There is narrowing...Anticoagulation has not been promoted in the treatment of intracranial arterial dissecting aneurysms due to the potential risk of mural hemorrhage, rupture

  16. Fast and easy phosphopeptide fractionation by combinatorial ERLIC-SCX solid-phase extraction for in-depth phosphoproteome analysis.

    PubMed

    Zarei, Mostafa; Sprenger, Adrian; Rackiewicz, Michal; Dengjel, Joern

    2016-01-01

    Mass spectrometry-based phosphoproteomic analysis is a powerful method for gaining a global, unbiased understanding of cellular signaling. Its accuracy and comprehensiveness stands or falls with the quality and choice of the applied phosphopeptide prefractionation strategy. This protocol covers a powerful but simple and rapid strategy for phosphopeptide prefractionation. The combinatorial use of two distinct chromatographic techniques that address the inverse physicochemical properties of peptides allows for superior fractionation efficiency of multiple phosphorylated peptides. In the first step, multiphosphorylated peptides are separated according to the number of negatively charged phosphosites by electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). A subsequent strong cation exchange (SCX) step separates mostly singly phosphorylated peptides in the ERLIC flow-through according to their positive charge. The presented strategy is inexpensive and adaptable to large and small amounts of starting material, and it allows highly multiplexed sample preparation. Because of its implementation as solid-phase extraction, the entire workflow takes only 2 h to complete.

  17. Extracellular Matrix Proteome and Phosphoproteome of Potato Reveals Functionally Distinct and Diverse Canonical and Non-Canonical Proteoforms

    PubMed Central

    Elagamey, Eman; Narula, Kanika; Sinha, Arunima; Aggarwal, Pooja Rani; Ghosh, Sudip; Chakraborty, Niranjan; Chakraborty, Subhra

    2016-01-01

    The extracellular matrix (ECM) has a molecular machinery composed of diverse proteins and proteoforms that combine properties of tensile strength with extensibility exhibiting growth-regulatory functions and self- and non-self-recognition. The identification of ECM proteoforms is the prerequisite towards a comprehensive understanding of biological functions accomplished by the outermost layer of the cell. Regulatory mechanisms of protein functions rely on post-translational modifications, phosphorylation in particular, affecting enzymatic activity, interaction, localization and stability. To investigate the ECM proteoforms, we have isolated the cell wall proteome and phosphoproteome of a tuberous crop, potato (Solanum tuberosum). LC-MS/MS analysis led to the identification of 38 proteins and 35 phosphoproteins of known and unknown functions. The findings may provide a better understanding of biochemical machinery and the integrated protein and phosphoprotein network of ECM for future functional studies of different developmental pathways and guidance cues in mechanosensing and integrity signaling. PMID:28248230

  18. Human R1441C LRRK2 regulates the synaptic vesicle proteome and phosphoproteome in a Drosophila model of Parkinson's disease.

    PubMed

    Islam, Md Shariful; Nolte, Hendrik; Jacob, Wright; Ziegler, Anna B; Pütz, Stefanie; Grosjean, Yael; Szczepanowska, Karolina; Trifunovic, Aleksandra; Braun, Thomas; Heumann, Hermann; Heumann, Rolf; Hovemann, Bernhard; Moore, Darren J; Krüger, Marcus

    2016-10-29

    Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset, autosomal dominant familial Parkinson's disease (PD) and variation at the LRRK2 locus contributes to the risk for idiopathic PD. LRRK2 can function as a protein kinase and mutations lead to increased kinase activity. To elucidate the pathophysiological mechanism of the R1441C mutation in the GTPase domain of LRRK2, we expressed human wild-type or R1441C LRRK2 in dopaminergic neurons of Drosophila and observe reduced locomotor activity, impaired survival and an age-dependent degeneration of dopaminergic neurons thereby creating a new PD-like model. To explore the function of LRRK2 variants in vivo, we performed mass spectrometry and quantified 3,616 proteins in the fly brain. We identify several differentially-expressed cytoskeletal, mitochondrial and synaptic vesicle proteins (SV), including synaptotagmin-1, syntaxin-1A and Rab3, in the brain of this LRRK2 fly model. In addition, a global phosphoproteome analysis reveals the enhanced phosphorylation of several SV proteins, including synaptojanin-1 (pThr1131) and the microtubule-associated protein futsch (pSer4106) in the brain of R1441C hLRRK2 flies. The direct phosphorylation of human synaptojanin-1 by R1441C hLRRK2 could further be confirmed by in vitro kinase assays. A protein-protein interaction screen in the fly brain confirms that LRRK2 robustly interacts with numerous SV proteins, including synaptojanin-1 and EndophilinA. Our proteomic, phosphoproteomic and interactome study in the Drosophila brain provides a systematic analyses of R1441C hLRRK2-induced pathobiological mechanisms in this model. We demonstrate for the first time that the R1441C mutation located within the LRRK2 GTPase domain induces the enhanced phosphorylation of SV proteins in the brain.

  19. Phosphoproteomes of Strongylocentrotus purpuratus shell and tooth matrix: identification of a major acidic sea urchin tooth phosphoprotein, phosphodontin

    PubMed Central

    2010-01-01

    Background Sea urchin is a major model organism for developmental biology and biomineralization research. However, identification of proteins involved in larval skeleton formation and mineralization processes in the embryo and adult, and the molecular characterization of such proteins, has just gained momentum with the sequencing of the Strongylocentrotus purpuratus genome and the introduction of high-throughput proteomics into the field. Results The present report contains the determination of test (shell) and tooth organic matrix phosphoproteomes. Altogether 34 phosphoproteins were identified in the biomineral organic matrices. Most phosphoproteins were specific for one compartment, only two were identified in both matrices. The sea urchin phosphoproteomes contained several obvious orthologs of mammalian proteins, such as a Src family tyrosine kinase, protein kinase C-delta 1, Dickkopf-1 and other signal transduction components, or nucleobindin. In most cases phosphorylation sites were conserved between sea urchin and mammalian proteins. However, the majority of phosphoproteins had no mammalian counterpart. The most interesting of the sea urchin-specific phosphoproteins, from the perspective of biomineralization research, was an abundant highly phosphorylated and very acidic tooth matrix protein composed of 35 very similar short sequence repeats, a predicted N-terminal secretion signal sequence, and an Asp-rich C-terminal motif, contained in [Glean3:18919]. Conclusions The 64 phosphorylation sites determined represent the most comprehensive list of experimentally identified sea urchin protein phosphorylation sites at present and are an important addition to the recently analyzed Strongylocentrotus purpuratus shell and tooth proteomes. The identified phosphoproteins included a major, highly phosphorylated protein, [Glean3:18919], for which we suggest the name phosphodontin. Although not sequence-related to such highly phosphorylated acidic mammalian dental

  20. Which Comes First: The Use of Computer Simulation of Frog Dissection or Conventional Dissection as Academic Exercise?

    ERIC Educational Resources Information Center

    Akpan, Joseph; Strayer, Jeremy

    2010-01-01

    Science educators and school administrators are reexamining the educational value of animal dissection in the nation's schools and are focusing on simulation as an instructional alternative. One implication of the debate is that simulations can lead to equivalent learning to hands-on dissection. The second implication is whether dissection is…

  1. Computed tomography: the investigation of choice for aortic dissection?

    PubMed Central

    Singh, H; Fitzgerald, E; Ruttley, M S

    1986-01-01

    Computed tomography has become established as complementary to aortography in the investigation of patients with suspected aortic dissection. Two cases of dissecting aneurysm are reported in which extensive aortography failed to show evidence of dissection. In both cases dissection was demonstrated by computed tomography. The diagnosis was confirmed in one case at operation and in the other case by follow up. It is suggested that computed tomography is the diagnostic method of first choice in aortic dissection. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:3730218

  2. Endovascular Treatment of Huge Dissecting Aneurysms Involving the Basilar Artery

    PubMed Central

    Yang, X.; Mu, S.; Lv, M.; Li, L.; Wu, Z.

    2007-01-01

    Summary Dissecting aneurysms involving the basilar artery (BA) are lesions with significant morbidity and mortality. Their management is controversial and often difficult. There is no generally approved strategy. Two cases of huge dissections involving the BA presented with subarachnoid hemorrhage in one case and mass effect in both cases. The dissection of case 1 involved the upper two thirds of the BA distal to the anterior inferior cerebellar arteries (AICA). Another dissection of case 2 involved the bilateral vertebral arteries (VA) distal to bilateral PICA and extended to upper third of the BA. After making a basket with coils inside the pseudoaneursym, proximal dissection was totally occluded in case 1. Dissection on the bilateral VA distal to the bilateral PICA and proximal BA was occluded in case 2 with a small residual dissection on the left VA. Case 1 had an excellent recovery with a durable image and clinical result. But recanalization and regrowth occurred in case 2, which might have originated from the residual dissection on the left VA, induced acute mass effect and sudden coma six weeks after the initial treatment. The residual and regrown dissection had to be occluded in a second intervention. The patient died two days later. BA occlusion is safe and efficient for dissections involving the BA as in our case and the literature. Proximal occlusion might be enough for huge and long lesions like ours. It seems that completely dense packing of proximal dissection is the key point to prevent recanalization. PMID:20566106

  3. Lymph Node Dissection for Differentiated Thyroid Cancer

    PubMed Central

    Mizrachi, Aviram; Shaha, Ashok R.

    2017-01-01

    Lymph node metastases in differentiated thyroid cancer (DTC) have a wide spectrum of clinical significance. Several variables are taken under consideration when trying to decide on the optimal management of patients with DTC. Routine prophylactic central and/or lateral lymph node dissection is not advocated with exception of central neck dissection for locally advanced tumors. When regarding recurrent disease, foundations have been laid for clinicians to make accurate decisions as to when to perform surgery and when to continue maintaining the patient’s disease under observation. These complex decisions are determined based upon multiple factors, not only regarding the patient’s disease but also the patient’s comprehension of the procedure and apprehension levels. Nevertheless if the patient and/or clinician are emotionally keen to surgically remove the disease then the procedure should be considered. PMID:28117285

  4. Counter traction makes endoscopic submucosal dissection easier.

    PubMed

    Oyama, Tsuneo

    2012-11-01

    Poor counter traction and poor field of vision make endoscopic submucosal dissection (ESD) difficult. Good counter traction allows dissections to be performed more quickly and safely. Position change, which utilizes gravity, is the simplest method to create a clear field of vision. It is useful especially for esophageal and colon ESD. The second easiest method is clip with line method. Counter traction made by clip with line accomplishes the creation of a clear field of vision and suitable counter traction thereby making ESD more efficient and safe. The author published this method in 2002. The name ESD was not established in those days; the name cutting endoscopic mucosal resection (EMR) or EMR with hook knife was used. The other traction methods such as external grasping forceps, internal traction, double channel scope, and double scopes method are introduced in this paper. A good strategy for creating counter traction makes ESD easier.

  5. [Open submucosal dissection: first case described].

    PubMed

    Portanova, Michel; Vesco, Eduardo; Morales, Domingo

    2007-01-01

    Endoscopic submucosal dissection is a new treatment, basically for the management of early gastric cancer, it is also a good option for large benign lesions if a "una pieza" resection needs to be performed. However this technique requires not only gastroenterologist with proven technical skill, but also some special devices not necessarily disposables in our country. The present paper describes the case of a patient with a large hyperplastic polyp located in the upper third of the stomach who underwent an open endoscopic submucosal dissection to resect the lesion owing to its size and characteristics. According our knowledge this is the first case in the medical literature describing the use of this technique during an open surgery.

  6. Spontaneous Coronary Artery Dissection: The Phantom Menace

    PubMed Central

    Spinthakis, Nikolaos; Abdulkareem, Nada; Farag, Mohamed; Gorog, Diana A.

    2016-01-01

    We present a case of a 66-year-old lady with chest pain, without dynamic 12-lead electrocardiographic (ECG) changes and normal serial troponin. Coronary angiography revealed a linear filing defect in the first obtuse marginal branch of the circumflex artery indicating coronary artery dissection, with superadded thrombus. She was managed medically with dual antiplatelet therapy and has responded well. Spontaneous coronary artery dissection (SCAD) is a rare cause of cardiac chest pain, which can be missed without coronary angiography. Unlike most other lesions in patients with unstable symptoms, where coronary intervention with stenting is recommended, patients with SCAD generally fare better with conservative measures than with intervention, unless there is hemodynamic instability. PMID:28197295

  7. Donors' attitudes towards body donation for dissection.

    PubMed

    Richardson, R; Hurwitz, B

    1995-07-29

    We report a survey in the UK of potential whole-body donors for dissection. 218 people (age range 19-97 years) answered a postal questionnaire, giving information about themselves, their reasons for donation, attitudes towards the dead body, funeral preferences and medical giving and receiving. In addition to altruism, motives included the wish to avoid funeral ceremonies, to avoid waste, and in a few cases, to evade the expense of a funeral. 44% understood that their bodies would be used as teaching material, 42% for experiments. Whilst 69% believed in one or more supernatural phenomena, only 39% said they were religious. 69% requested cremation after dissection; 2% wanted to be buried. The notion of money incentives to promote donation was overwhelmingly rejected.

  8. Nested Dissection Interface Reconstruction in Pececillo

    SciTech Connect

    Jibben, Zechariah Joel; Carlson, Neil N.; Francois, Marianne M.

    2016-09-13

    A nested dissection method for interface reconstruction in a volume tracking framework has been implemented in Pececillo, a mini-app for Truchas, which is the ASC code for casting and additive manufacturing. This method provides a significant improvement over the traditional onion-skin method, which does not appropriately handle T-shaped multimaterial intersections and dynamic contact lines present in additive manufacturing simulations. The resulting implementation lays the groundwork for further research in contact angle estimates and surface tension calculations.

  9. Building mental models by dissecting physical models.

    PubMed

    Srivastava, Anveshna

    2016-01-01

    When students build physical models from prefabricated components to learn about model systems, there is an implicit trade-off between the physical degrees of freedom in building the model and the intensity of instructor supervision needed. Models that are too flexible, permitting multiple possible constructions require greater supervision to ensure focused learning; models that are too constrained require less supervision, but can be constructed mechanically, with little to no conceptual engagement. We propose "model-dissection" as an alternative to "model-building," whereby instructors could make efficient use of supervisory resources, while simultaneously promoting focused learning. We report empirical results from a study conducted with biology undergraduate students, where we demonstrate that asking them to "dissect" out specific conceptual structures from an already built 3D physical model leads to a significant improvement in performance than asking them to build the 3D model from simpler components. Using questionnaires to measure understanding both before and after model-based interventions for two cohorts of students, we find that both the "builders" and the "dissectors" improve in the post-test, but it is the latter group who show statistically significant improvement. These results, in addition to the intrinsic time-efficiency of "model dissection," suggest that it could be a valuable pedagogical tool.

  10. Future directions of duodenal endoscopic submucosal dissection

    PubMed Central

    Matsumoto, Satohiro; Miyatani, Hiroyuki; Yoshida, Yukio

    2015-01-01

    Endoscopic therapies for lesions of the duodenum are technically more difficult than those for lesions of the other parts of the gastrointestinal tract due to the anatomical features of the duodenum, and the incidence rate of complications such as perforation and bleeding is also higher. These aforementioned trends were especially noticeable for the case of duodenal endoscopic submucosal dissection (ESD). The indication for ESD of duodenal tumors should be determined by assessment of the histopathology, macroscopic morphology, and diameter of the tumors. The three types of candidate lesions for endoscopic therapy are adenoma, carcinoma, and neuroendocrine tumors. For applying endoscopic therapies to duodenal lesions, accurate preoperative histopathological diagnosis is necessary. The most important technical issue in duodenal ESD is the submucosal dissection process. In duodenal ESD, a short needle-type knife is suitable for the mucosal incision and submucosal dissection processes, and the Small-caliber-tip Transparent hood is an important tool. After endoscopic therapies, the wound should be closed by clipping in order to prevent complications such as secondary hemorrhage and delayed perforation. At present, the criteria for selection between ESD and EMR vary among institutions. The indications for ESD should be carefully considered. Duodenal ESD should have limitations, such as the need for its being performed by experts with abundant experience in performing the procedure. PMID:25901218

  11. Whole vitreous humor dissection for vitreodynamic analysis.

    PubMed

    Murali, Karthik; Kashani, Amir H; Humayun, Mark S

    2015-05-24

    The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous studies have shown the results of such dissections, the detailed steps have not been described, precluding researchers outside the field from replicating their methods. Other studies harvest vitreous either through aspiration, which does not maintain the vitreous structure anatomy, or through partial dissection, which only isolates the vitreous core. The proposed method isolates the whole vitreous body, with the vitreous core and cortex intact, while maintaining vitreous anatomy and structural integrity. In this method, a full thickness scleral flap in an enucleated porcine eye is first created and through this, the choroid tissue can be separated from the sclera. The scleral flap is then expanded and the choroid is completely separated from the sclera. Finally the choroid-retina tissue is peeled off the vitreous to leave an isolated intact vitreous body. The proposed vitreous dissection technique can be used to study physical properties of the vitreous humor. In particular, this method has significance for experimental studies involving drug delivery, vitreo-retinal oxygen transport, and intraocular convection.

  12. Student attitudes to whole body donation are influenced by dissection.

    PubMed

    Cahill, Kevin C; Ettarh, Raj R

    2008-01-01

    Given the important role that anatomical dissection plays in the shaping of medical student attitudes to life and death, these attitudes have not been evaluated in the context of whole body donation for medical science. First year students of anatomy in an Irish university medical school were surveyed by questionnaire before and after the initial dissection and again after 9 weeks of anatomical dissection. Analysis of student responses to the idea of whole body donation by an unrelated stranger, a family member, or by the respondent showed that a priori attitudes to donation by a stranger did not change with exposure to dissection. However, student opposition to donation by a family member was evident immediately after the initial dissection and was sustained throughout the duration of this study. Support for the idea of donating their bodies to medical science decreased significantly among respondents after exposure to dissection (31.5% before dissection, 19.6% after dissecting for 9 weeks) but not to levels reported in the general population in other studies. This study demonstrates that where dissection forms a part of anatomy teaching, students expect to learn anatomy by dissecting donors whom they do not know. As a potential donor population, students are reluctant to become emotionally involved in the donation process and are unwilling to become donors themselves.

  13. Peer-Assisted Learning in a Gross Anatomy Dissection Course.

    PubMed

    Han, Eui-Ryoung; Chung, Eun-Kyung; Nam, Kwang-Il

    2015-01-01

    Peer-assisted learning encourages students to participate more actively in the dissection process and promotes thoughtful dissection. We implemented peer-assisted dissection in 2012 and compared its effects on students' self-assessments of learning and their academic achievement with those of faculty-led dissection. All subjects performed dissections after a lecture about upper-limb gross anatomy. Experimental group (n = 134) dissected a cadaver while guided by peer tutors who had prepared for the dissection in advance, and control group (n = 71) dissected a cadaver after the introduction by a faculty via prosection. Self-assessment scores regarding the learning objectives related to upper limbs were significantly higher in experimental group than in control group. Additionally, experimental group received significantly higher academic scores than did control group. The students in peer-assisted learning perceived themselves as having a better understanding of course content and achieved better academic results compared with those who participated in faculty-led dissection. Peer-assisted dissection contributed to self-perception and to the ability to retain and explain anatomical knowledge.

  14. Extracranial and intracranial vertebrobasilar dissections: diagnosis and prognosis

    PubMed Central

    de Bray, J M; Penisson-Besnier, I; Dubas, F; Emile, J

    1997-01-01

    OBJECTIVES—To compare the diagnosis and prognosis of extracranial versus intracranial vertebral artery dissections without intracerebral haemorrhage.
METHODS—Twenty two vertebral artery dissections were defined by intra-arterial angiography and classified in two groups: group 1,nine extracranial dissections (seven patients) and group 2, 13intracranial dissections (nine patients), involving the basilar artery in five cases. Bilateral dissections were found in 38% of the population. Before angiography, all the patients had been investigated by continuous wave Doppler, colour coded Doppler, and transcranial Doppler. Mean follow up was 44months.
RESULTS—The two most important symptoms of both dissections (81% of patients) were unbearable pain preceding stroke and progressive onset of stroke within a few hours. Severe ultrasonic abnormalities were present in 94% of the patients whereas specific ultrasonic signs (segmental dilation with eccentric channel) were rare (19%) in both groups. Major strokes and brainstem strokes represented respectively 67% and 78% in intracranial versus 43% and 29% in extracranial dissections. Severe sequelae (permanent disabling motor or cerebellar deficit) were more often associated with intracranial (44%) than with extracranial dissections (14%). No recurrence of dissection and no cerebral haemorrhage were found under heparin. Significant factors of poor outcome (P< 0.05) were the initial severity of the stroke and the bilateral location of dissections.
CONCLUSION—The combination of a pain and a progressive onset of the stroke, corroborated by ultrasonic findings, could have helped to recognise most of these types of dissections. Intracranial dissections have a poorer prognosis than extracranial dissections.

 PMID:9221967

  15. Background music in the dissection laboratory: impact on stress associated with the dissection experience.

    PubMed

    Anyanwu, Emeka G

    2015-06-01

    Notable challenges, such as mental distress, boredom, negative moods, and attitudes, have been associated with learning in the cadaver dissection laboratory (CDL). The ability of background music (BM) to enhance the cognitive abilities of students is well documented. The present study was designed to investigate the impact of BM in the CDL and on stress associated with the dissection experience. After 8 wk of normal dissection without BM, various genres of BM were introduced into the cadaver dissection sessions of 260 medical and dental students for 3 wk. Feedback on the impact of BM on students in the CDL and students' attitude were accessed using a questionnaire. Psychological stress assessment was done using Psychological Stress Measure 9. Two batches of 30 students each were made to dissect same areas of the body for 2 h, one batch with BM playing and the other batch without. The same examination was given to both groups at the end. Over 90% of the participants expressed a desire to incorporate BM into the CDL; 87% of the sampled population that expressed love for music also reported BM to be a very useful tool that could be used to enhance learning conditions in the CDL. A strong positive relationship was established between love for music and its perception as a tool for learning in the CDL (P < 0.001). Students that studied under the influence of BM had significantly higher scores (P < 0.001) in the overall examination result. BM reduced the level of stress associated with the dissection experience by ∼33%.

  16. Laser micro-dissection and qPCR for identifying specific HPV types responsible for malignancy in penile lesions.

    PubMed

    Lebelo, Ramokone L; Thys, Sofie; Benoy, Ina; Depuydt, Christophe E; Bogers, John-Paul; Bida, Meshack N; Mphahlele, M Jeffrey

    2015-10-01

    The aim of the study was to identify specific human papillomavirus (HPV) type responsible for malignancy in penile tissue samples using laser micro-dissection and TaqMan quantitative real-time PCR (qPCR). The study was based on two pre-malignant and seven malignant penile tissue samples and laser micro-dissection was performed on all. Genotyping was performed on whole tissue sections and laser micro-dissection samples using qPCR. Two whole tissue section samples were HPV negative while seven were HPV positive. In four samples that were single HPV infections with whole tissue section PCR, identical HPV types were confirmed with laser micro-dissection PCR. Clearly confirming that the single HPV type detected is responsible for malignancy. In two samples that had multiple HPV infections with whole tissue section PCR, only one HPV type with the highest viral load was detected with laser micro-dissection PCR, suggesting that the HPV type with the highest viral load is most likely the cause of that particular lesion. HPV 11 and/or HPV 16 were the only types detected with laser micro-dissection PCR in these cases, compared to multiple HPV types (HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, and HPV 39) initially detected with whole tissue section PCR. HPV 11 was associated with verrucous lesions while HPV 16 was associated with squamous cell carcinoma and PIN 3 lesions. This study confirms that laser micro-dissection and qPCR are essential tools in identifying the HPV types responsible for malignancy in penile lesions, particularly in samples with multiple infections.

  17. Endoscopic submucosal dissection for early esophageal cancer associated with achalasia.

    PubMed

    Ohkura, Yu; Iizuka, Toshiro; Kikuchi, Daisuke; Yamashita, Satoshi; Nakamura, Masanori; Matsui, Akira; Mitani, Toshifumi; Hoteya, Shu; Kaise, Mitsuru; Yahagi, Naohisa

    2013-01-01

    Esophageal achalasia is often associated with esophageal cancer. However, in many cases, esophageal cancer tends to be found in an advanced stage, with a poor prognosis. However, early-stage cancer was detected recently due to the advances in endoscopic instruments. In those cases, it is important to facilitate successful treatment by endoscopic submucosal dissection. We analyzed a total of six cases of esophageal cancer with achalasia in four patients treated with endoscopic submucosal dissection. Three features common to all six cases had a bearing on how endoscopic submucosal dissection was performed. First, esophageal dilatation and diminished peristalsis facilitated the performance of successful endoscopic submucosal dissection. Second, the esophageal wall was thickened, primarily with muscular tissue. Third, the submucosal layer contained abundant blood vessels that made it difficult to minimize bleeding during dissection. Those findings suggest that endoscopic submucosal dissection for early esophageal cancer associated with achalasia is a safe and potentially curative procedure. It is important, therefore, to detect esophageal cancer early.

  18. Treatment of Vertebro-Basilar Dissecting Aneurysms Using Intravascular Stents

    PubMed Central

    Yamasaki, S.; Hashimoto, K.; Kawano, Y.; Yoshimura, M.; Yamamoto, T.; Hara, M.

    2006-01-01

    Summary Endovascular surgery is an established primary therapeutic modality for dissecting aneurysms at vertebro-basilar arteries. Intravascular stents can be used to treat the dissecting aneurysms for which simple obliteration procedures cannot be used. In such cases, stent implantation alone or a combination of stents and coils need to be selected properly by taking into consideration the site and shape of dissections. In this report, three patterns of stent application are described and their method of selection is discussed. PMID:20569619

  19. Spontaneous Coronary Dissection Masquerading as Benign Fascicular Ventricular Tachycardia.

    PubMed

    Ho, Sara Wei-Fen; Lin, Weiqin; Chan, Koo Hui; Seow, Swee-Chong

    2016-01-01

    Spontaneous coronary artery dissection is an uncommon cause of acute coronary syndrome. Diagnosis of coronary artery dissection is made on coronary angiogram and prompt revascularisation is the key in management. We present a case of coronary artery dissection with an atypical presentation of cardiac arrhythmia mimicking benign fascicular ventricular tachycardia. A high index of suspicion and early coronary angiogram allowed us to diagnose and treat this potentially life-threatening disease.

  20. Doing Dissections Differently: A Structured, Peer-Assisted Learning Approach to Maximizing Learning in Dissections

    ERIC Educational Resources Information Center

    Hall, Emma R.; Davis, Rachel C.; Weller, Renate; Powney, Sonya; Williams, Sarah B.

    2013-01-01

    Areas of difficulty faced by our veterinary medicine students, with respect to their learning in dissection classes, were identified. These challenges were both general adult-learning related and specific to the discipline of anatomy. Our aim was to design, implement, and evaluate a modified reciprocal peer-assisted/team-based learning…

  1. Background Music in the Dissection Laboratory: Impact on Stress Associated with the Dissection Experience

    ERIC Educational Resources Information Center

    Anyanwu, Emeka G.

    2015-01-01

    Notable challenges, such as mental distress, boredom, negative moods, and attitudes, have been associated with learning in the cadaver dissection laboratory (CDL). The ability of background music (BM) to enhance the cognitive abilities of students is well documented. The present study was designed to investigate the impact of BM in the CDL and on…

  2. Dissection and Dissection-Associated Required Experiences Improve Student Performance in Gross Anatomy: Differences among Quartiles

    ERIC Educational Resources Information Center

    Pizzimenti, Marc A.; Pantazis, Nicholas; Sandra, Alexander; Hoffmann, Darren S.; Lenoch, Susan; Ferguson, Kristi J.

    2016-01-01

    To promote student learning, educational strategies should provide multiple levels of engagement with the subject matter. This study investigated examination data from five first year medical gross anatomy class cohorts (692 students) to determine if enhanced student performance was correlated with learning through dissection in a course that used…

  3. Dissection videos do not improve anatomy examination scores.

    PubMed

    Mahmud, Waqas; Hyder, Omar; Butt, Jamaal; Aftab, Arsalan

    2011-01-01

    In this quasi-experimental study, we describe the effect of showing dissection videos on first-year medical students' performance in terms of test scores during a gross anatomy course. We also surveyed students' perception regarding the showing of dissection videos. Two hundred eighty-seven first-year medical students at Rawalpindi Medical College in Pakistan, divided into two groups, dissected one limb in first term and switched over to the other limb in the second term. During the second term, instruction was supplemented by dissection videos. Second-term anatomy examination marks were compared with first-term scores and with results from first-year medical students in previous years. Multiple linear regression analysis was performed, with term scores (continuous, 0-200) as the dependent variable. Students shown dissection videos scored 1.26 marks higher than those not shown. The relationship was not statistically significant (95% CI: -1.11, 3.70; P = 0.314). Ninety-three percent of students favored regular inclusion of dissection videos in curriculum, and 50% termed it the best source for learning gross anatomy. Seventy-six percent of students did not perform regular cadaver dissection. The most frequent reason cited for not performing regular dissection was high student-cadaver ratio. Dissection videos did not improve performance on final examination scores; however, students favored their use.

  4. Acute aortic dissection provoked by sneeze: a case report.

    PubMed

    Baydin, A; Nural, M S; Güven, H; Deniz, T; Bildik, F; Karaduman, A

    2005-10-01

    The response of the abdominal viscera and the contraction of the intercostal muscles during the respiratory phase of sneezing increases intrathoracic pressure, which may lead to several complications. However, there are no reports in the literature concerning aortic dissection after sneezing. We report a patient in whom the development of dissection was secondary to sneezing, although hypertension was present as a risk factor, and we discuss the relationship between sneezing and aortic dissection. To our knowledge, this is the first report of aortic dissection provoked by sneezing in the literature.

  5. Orientation to dissection: Assisting students through the transition.

    PubMed

    Lamdin, R; Weller, J; Kerse, N

    2012-03-01

    Human dissection continues to be strongly argued for teaching human anatomy to medical students and is technically and emotionally demanding. An orientation to dissection and the laboratory are provided for students before beginning their work because students' and families' reactions to dissection are often complex. This study explored medical students' experiences of attending an orientation to human dissection and the anatomy laboratory. Students' reactions, feelings, and thoughts were enquired about 1 year after beginning dissection at the University of Auckland, New Zealand. Qualitative research methods, specifically one-on-one semistructured interview were utilized. Third-year medical students self-selected into the study and were interviewed 1 year after entering the laboratory. Transcribed audiotapes of the interviews were analyzed for themes across the interviews. One year after dissection students have vivid memories with differing ways of viewing the body that may help or hinder with dissection. The themes presented include orientation, student anticipation, psychological approach to the body, normalizing-continuing disquiet, and social reference. The orientation eases student entry into the laboratory. There can be ongoing feelings of ambivalence regards the body for some students. Novel findings include that students not only have their own feelings to deal with but also those of friends and family who question them and may feel uncomfortable with the idea of them dissecting. Even one year after beginning dissection, students may emotionally struggle with their work and may require further support, including how they talk about sensitive topics with other people.

  6. The Effect of a Prior Dissection Simulation on Middle School Students' Dissection Performance and Understanding of the Anatomy and Morphology of the Frog

    NASA Astrophysics Data System (ADS)

    Akpan, Joseph Paul; Andre, Thomas

    1999-06-01

    Science teachers, school administrators, educators, and the scientific community are faced with ethical controversies over animal dissection in classrooms. Simulation has been proposed as a way of dealing with this issue. One intriguing previous finding was that use of an interactive videodisc dissection facilitated performance on a subsequent actual dissection. This study examined the prior use of simulation of frog dissection in improving students' actual dissection performance and learning of frog anatomy and morphology. There were three experimental conditions: simulation before dissection (SBD); dissection before simulation (DBS); or dissection-only (DO). Results of the study indicated that students receiving SBD performed significantly better than students receiving DBS or DO on both actual dissection and knowledge of the anatomy and morphology. Students' attitudes toward the use of animals for dissection did not change significantly from pretest to posttest and did not interact with treatment. The genders did not differ in achievement, but males were more favorable towards dissection and computers than were females.

  7. Laser versus dissection technique of tonsillectomy.

    PubMed

    Ishlah, L Wan; Fahmi, A M; Srinovianti, N

    2005-03-01

    Tonsillectomy is the single most common operation performed in Ear Nose and Throat Department. Various methods of tonsillectomy have been practiced over the century aimed at reducing or eliminating intraoperative and postoperative morbidity. Due to various blood supplies received, intraoperative bleeding is the most difficult problem and securing it is time-consuming. The time taken to control the bleeding would invariably determine the length of operation. Common postoperative complications are bleeding and pain. This study evaluated the operative time, intraoperative blood loss, postoperative pain and other postoperative complications of tonsillectomy performed by laser as compared to conventional dissection technique. This is a prospective randomized study whereby sixty patients were divided into two groups of equal number. In one group, the tonsillectomy performed by laser and in the other group the tonsillectomy performed by conventional dissection technique. Operative time and amount of blood loss is significantly reduced in the laser group. Total postoperative pain and post operative complications were not significantly different between the two groups. Tonsillectomy by using laser have shown less intraoperative bleeding and shortened the operative time. In the hospital where laser machine and expertise are available, it is justifiable to use this technique as effective method of performing tonsillectomy.

  8. Homarus Americanus Stomatogastric Nervous System Dissection

    PubMed Central

    Tobin, Anne-Elise; Bierman, Hilary S.

    2009-01-01

    With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the activity of interest and are accessible and amenable to experimental methods. The stomatogastric nervous system (STNS) of the American lobster (Homarus americanus; also know was the Atlantic or Maine lobster) has been established as a model system for studying rhythm generating networks and neuromodulation of networks. The STNS consists of 3 anterior ganglia (2 commissural ganglia and an oesophageal ganglion), containing modulatory neurons that project centrally to the stomatogastric ganglion (STG). The STG contains approximately 30 neurons that comprise two central pattern generating networks, the pyloric and gastric networks that underlie feeding behaviors in crustaceans1,2. While it is possible to study this system in vivo3, the STNS continues to produce its rhythmic activity when isolated in vitro. Physical isolation of the STNS in a dish allows for easy access to the somata in the ganglia for intracellular electrophysiological recordings and to the nerves of the STNS for extracellular recordings. Isolating the STNS is a two-part process. The first part, dissecting the stomach from the animal, is described in an accompanying video article4. In this video article, fine dissection techniques are used to isolate the STNS from the stomach. This procedure results in a nervous system preparation that is available for electrophysiological recordings. PMID:19483669

  9. Genetic Dissection of a QTL Affecting Bone Geometry

    PubMed Central

    Sabik, Olivia L.; Medrano, Juan F.; Farber, Charles R.

    2017-01-01

    Parameters of bone geometry such as width, length, and cross-sectional area are major determinants of bone strength. Although these traits are highly heritable, few genes influencing bone geometry have been identified. Here, we dissect a major quantitative trait locus (QTL) influencing femur size. This QTL was originally identified in an F2 cross between the C57BL/6J-hg/hg (HG) and CAST/EiJ strains and was referred to as femur length in high growth mice 2 (Feml2). Feml2 was located on chromosome (Chr.) 9 at ∼20 cM. Here, we show that the HG.CAST-(D9Mit249-D9Mit133)/Ucd congenic strain captures Feml2. In an F2 congenic cross, we fine-mapped the location of Feml2 to an ∼6 Mbp region extending from 57.3 to 63.3 Mbp on Chr. 9. We have identified candidates by mining the complete genome sequence of CAST/EiJ and through allele-specific expression (ASE) analysis of growth plates in C57BL/6J × CAST/EiJ F1 hybrids. Interestingly, we also find that the refined location of Feml2 overlaps a cluster of six independent genome-wide associations for human height. This work provides the foundation for the identification of novel genes affecting bone geometry. PMID:28082324

  10. Functional dissection of Odorant binding protein genes in Drosophila melanogaster

    PubMed Central

    Swarup, S; Williams, T I; Anholt, R R H

    2011-01-01

    Most organisms rely on olfaction for survival and reproduction. The olfactory system of Drosophila melanogaster is one of the best characterized chemosensory systems and serves as a prototype for understanding insect olfaction. Olfaction in Drosophila is mediated by multigene families of odorant receptors and odorant binding proteins (OBPs). Although molecular response profiles of odorant receptors have been well documented, the contributions of OBPs to olfactory behavior remain largely unknown. Here, we used RNAi-mediated suppression of Obp gene expression and measurements of behavioral responses to 16 ecologically relevant odorants to systematically dissect the functions of 17 OBPs. We quantified the effectiveness of RNAi-mediated suppression by quantitative real-time polymerase chain reaction and used a proteomic liquid chromatography and tandem mass spectrometry procedure to show target-specific suppression of OBPs expressed in the antennae. Flies in which expression of a specific OBP is suppressed often show altered behavioral responses to more than one, but not all, odorants, in a sex-dependent manner. Similarly, responses to a specific odorant are frequently affected by suppression of expression of multiple, but not all, OBPs. These results show that OBPs are essential for mediating olfactory behavioral responses and suggest that OBP-dependent odorant recognition is combinatorial. PMID:21605338

  11. Large-scale zebrafish embryonic heart dissection for transcriptional analysis.

    PubMed

    Lombardo, Verónica A; Otten, Cécile; Abdelilah-Seyfried, Salim

    2015-01-12

    The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

  12. Genetic Dissection of a QTL Affecting Bone Geometry.

    PubMed

    Sabik, Olivia L; Medrano, Juan F; Farber, Charles R

    2017-03-10

    Parameters of bone geometry such as width, length, and cross-sectional area are major determinants of bone strength. Although these traits are highly heritable, few genes influencing bone geometry have been identified. Here, we dissect a major quantitative trait locus (QTL) influencing femur size. This QTL was originally identified in an F2 cross between the C57BL/6J-hg/hg (HG) and CAST/EiJ strains and was referred to as femur length in high growth mice 2 (Feml2). Feml2 was located on chromosome (Chr.) 9 at ∼20 cM. Here, we show that the HG.CAST-(D9Mit249-D9Mit133)/Ucd congenic strain captures Feml2 In an F2 congenic cross, we fine-mapped the location of Feml2 to an ∼6 Mbp region extending from 57.3 to 63.3 Mbp on Chr. 9. We have identified candidates by mining the complete genome sequence of CAST/EiJ and through allele-specific expression (ASE) analysis of growth plates in C57BL/6J × CAST/EiJ F1 hybrids. Interestingly, we also find that the refined location of Feml2 overlaps a cluster of six independent genome-wide associations for human height. This work provides the foundation for the identification of novel genes affecting bone geometry.

  13. Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: Quercus ilex L. subsp. ballota [Desf.] Samp.

    PubMed Central

    Romero-Rodríguez, M. Cristina; Abril, Nieves; Sánchez-Lucas, Rosa; Jorrín-Novo, Jesús V.

    2015-01-01

    As a preliminary step in the phosphoproteome analysis of germinating seeds (0 and 24 h after seed imbibition) and early grown seedlings (216 h after seed imbibition) from a non-orthodox sp. Quercus ilex, a multiplex (SYPRO-Ruby and Pro-Q DPS) staining of high-resolution 2-DE gels was used. By using this protocol it was possible to detect changes in protein-abundance and/or phosphorylation status. This simple approach could be a good complementary alternative to the enrichment protocols used in the search for phosphoprotein candidates. While 482 spots were visualized with SYPRO-Ruby, 222 were with Pro-Q DPS. Statistically significant differences in spot intensity were observed among samples, these corresponding to 85 SYPRO-Ruby-, 20 Pro-Q-DPS-, and 35 SYPRO-Ruby and Pro-Q-DPS-stained spots. Fifty-five phosphoprotein candidates showing qualitative or quantitative differences between samples were subjected to MALDI-TOF-TOF MS analysis, with 20 of them being identified. Identified proteins belonged to five different functional categories, namely: carbohydrate and amino acid metabolism, defense, protein folding, and oxidation-reduction processes. With the exception of a putative cyclase, the other 19 proteins had at least one orthologous phosphoprotein in Arabidopsis thaliana, Medicago truncatula, N. tabacum, and Glycine max. Out of the 20 identified, seven showed differences in intensity in Pro-Q-DPS but not in SYPRO-Ruby-stained gels, including enzymes of the glycolysis and amino acid metabolism. This bears out that theory the regulation of these enzymes occurs at the post-translational level by phosphorylation with no changes at the transcriptional or translational level. This is different from the mechanism reported in orthodox seeds, in which concomitant changes in abundance and phosphorylation status have been observed for these enzymes. PMID:26322061

  14. Comparative phosphoproteome profiling reveals a function of the STN8 kinase in fine-tuning of cyclic electron flow (CEF)

    PubMed Central

    Reiland, Sonja; Finazzi, Giovanni; Endler, Anne; Willig, Adrian; Baerenfaller, Katja; Grossmann, Jonas; Gerrits, Bertran; Rutishauser, Dorothea; Gruissem, Wilhelm; Rochaix, Jean-David; Baginsky, Sacha

    2011-01-01

    Important aspects of photosynthetic electron transport efficiency in chloroplasts are controlled by protein phosphorylation. Two thylakoid-associated kinases, STN7 and STN8, have distinct roles in short- and long-term photosynthetic acclimation to changes in light quality and quantity. Although some substrates of STN7 and STN8 are known, the complexity of this regulatory kinase system implies that currently unknown substrates connect photosynthetic performance with the regulation of metabolic and regulatory functions. We performed an unbiased phosphoproteome-wide screen with Arabidopsis WT and stn8 mutant plants to identify unique STN8 targets. The phosphorylation status of STN7 was not affected in stn8, indicating that kinases other than STN8 phosphorylate STN7 under standard growth conditions. Among several putative STN8 substrates, PGRL1-A is of particular importance because of its possible role in the modulation of cyclic electron transfer. The STN8 phosphorylation site on PGRL1-A is absent in both monocotyledonous plants and algae. In dicots, spectroscopic measurements with Arabidopsis WT, stn7, stn8, and stn7/stn8 double-mutant plants indicate a STN8-mediated slowing down of the transition from cyclic to linear electron flow at the onset of illumination. This finding suggests a possible link between protein phosphorylation by STN8 and fine-tuning of cyclic electron flow during this critical step of photosynthesis, when the carbon assimilation is not commensurate to the electron flow capacity of the chloroplast. PMID:21768351

  15. Phosphoproteomics reveals resveratrol-dependent inhibition of Akt/mTORC1/S6K1 signaling.

    PubMed

    Alayev, Anya; Doubleday, Peter F; Berger, Sara Malka; Ballif, Bryan A; Holz, Marina K

    2014-12-05

    Resveratrol, a plant-derived polyphenol, regulates many cellular processes, including cell proliferation, aging and autophagy. However, the molecular mechanisms of resveratrol action in cells are not completely understood. Intriguingly, resveratrol treatment of cells growing in nutrient-rich conditions induces autophagy, while acute resveratrol treatment of cells in a serum-deprived state inhibits autophagy. In this study, we performed a phosphoproteomic analysis after applying resveratrol to serum-starved cells with the goal of identifying the acute signaling events initiated by resveratrol in a serum-deprived state. We determined that resveratrol in serum-starved conditions reduces the phosphorylation of several proteins belonging to the mTORC1 signaling pathway, most significantly, PRAS40 at T246 and S183. Under these same conditions, we also found that resveratrol altered the phosphorylation of several proteins involved in various biological processes, most notably transcriptional modulators, represented by p53, FOXA1, and AATF. Together these data provide a more comprehensive view of both the spectrum of phosphoproteins upon which resveratrol acts as well as the potential mechanisms by which it inhibits autophagy in serum-deprived cells.

  16. Large-Scale Phosphoproteome of Human Whole Saliva Using Disulphide-Thiol-Interchange Covalent Chromatography and Mass Spectrometry

    PubMed Central

    Salih, Erdjan; Siqueira, Walter L.; Helmerhorst, Eva J.; Oppenheim, Frank G.

    2010-01-01

    Thus far only a handful of phosphoproteins with important biological functions have been identified and characterized in oral fluids and these include some of the abundant protein constituents of saliva. Whole saliva (WS) samples were trypsin digested followed by chemical derivatization using dithiothreitol (DTT) of the phospho-serine/threonine containing peptides. The DTT-phosphopeptides were enriched by covalent disulphide-thiol-interchange chromatography and analysis by nano-flow LC-ESI-MS/MS. The specificity of DTT chemical derivatization was evaluated separately under different base-catalyzed conditions with NaOH and Ba(OH)2, blocking cysteine residues by iodoacetamide and enzymatic O-deglycosylation prior to DTT reaction. Further analysis of WS samples which were subjected to either of these conditions provided supporting evidence for phosphoprotein identifications. The combined chemical strategies and mass spectrometric analyses identified 65 phosphoproteins in WS of which 28 were based on two or more peptide identification criteria with high confidence, and 37 were based on a single phosphopeptide identification. Most of the identified proteins, ~80%, were hitherto unknown phosphoprotein components. This study represents the first large-scale documentation of phosphoproteins of WS. The origins and identity of WS phosphoproteome suggest significant implications for both basic science and the development of novel biomarkers/diagnostic tools for both systemic and oral disease states. PMID:20659418

  17. Comparative Phosphoproteomics Analysis of VEGF and Angiopoietin-1 Signaling Reveals ZO-1 as a Critical Regulator of Endothelial Cell Proliferation.

    PubMed

    Chidiac, Rony; Zhang, Ying; Tessier, Sylvain; Faubert, Denis; Delisle, Chantal; Gratton, Jean-Philippe

    2016-05-01

    VEGF and angiopoietin-1 (Ang-1) are essential factors to promote angiogenesis through regulation of a plethora of signaling events in endothelial cells (ECs). Although pathways activated by VEGF and Ang-1 are being established, the unique signaling nodes conferring specific responses to each factor remain poorly defined. Thus, we conducted a large-scale comparative phosphoproteomic analysis of signaling pathways activated by VEGF and Ang-1 in ECs using mass spectrometry. Analysis of VEGF and Ang-1 networks of regulated phosphoproteins revealed that the junctional proteins ZO-1, ZO-2, JUP and p120-catenin are part of a cluster of proteins phosphorylated following VEGF stimulation that are linked to MAPK1 activation. Down-regulation of these junctional proteins led to MAPK1 activation and accordingly, increased proliferation of ECs stimulated specifically by VEGF, but not by Ang-1. We identified ZO-1 as the central regulator of this effect and showed that modulation of cellular ZO-1 levels is necessary for EC proliferation during vascular development of the mouse postnatal retina. In conclusion, we uncovered ZO-1 as part of a signaling node activated by VEGF, but not Ang-1, that specifically modulates EC proliferation during angiogenesis.

  18. Phosphoproteome profiles of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea during exponential growth in axenic cultures.

    PubMed

    Davanture, Marlène; Dumur, Jérôme; Bataillé-Simoneau, Nelly; Campion, Claire; Valot, Benoît; Zivy, Michel; Simoneau, Philippe; Fillinger, Sabine

    2014-07-01

    This study describes the gel-free phosphoproteomic analysis of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea grown in vitro under nonlimiting conditions. Using a combination of strong cation exchange and IMAC prior to LC-MS, we identified over 1350 phosphopeptides per fungus representing over 800 phosphoproteins. The preferred phosphorylation sites were found on serine (>80%) and threonine (>15%), whereas phosphorylated tyrosine residues were found at less than 1% in A. brassicicola and at a slightly higher ratio in B. cinerea (1.5%). Biological processes represented principally among the phoshoproteins were those involved in response and transduction of stimuli as well as in regulation of cellular and metabolic processes. Most known elements of signal transduction were found in the datasets of both fungi. This study also revealed unexpected phosphorylation sites in histidine kinases, a category overrepresented in filamentous ascomycetes compared to yeast. The data have been deposited to the ProteomeXchange database with identifier PXD000817 (http://proteomecentral.proteomexchange.org/dataset/PXD000817).

  19. Phosphoproteomics Reveals Resveratrol-Dependent Inhibition of Akt/mTORC1/S6K1 Signaling

    PubMed Central

    2015-01-01

    Resveratrol, a plant-derived polyphenol, regulates many cellular processes, including cell proliferation, aging and autophagy. However, the molecular mechanisms of resveratrol action in cells are not completely understood. Intriguingly, resveratrol treatment of cells growing in nutrient-rich conditions induces autophagy, while acute resveratrol treatment of cells in a serum-deprived state inhibits autophagy. In this study, we performed a phosphoproteomic analysis after applying resveratrol to serum-starved cells with the goal of identifying the acute signaling events initiated by resveratrol in a serum-deprived state. We determined that resveratrol in serum-starved conditions reduces the phosphorylation of several proteins belonging to the mTORC1 signaling pathway, most significantly, PRAS40 at T246 and S183. Under these same conditions, we also found that resveratrol altered the phosphorylation of several proteins involved in various biological processes, most notably transcriptional modulators, represented by p53, FOXA1, and AATF. Together these data provide a more comprehensive view of both the spectrum of phosphoproteins upon which resveratrol acts as well as the potential mechanisms by which it inhibits autophagy in serum-deprived cells. PMID:25311616

  20. PAPE (Prefractionation-Assisted Phosphoprotein Enrichment): A Novel Approach for Phosphoproteomic Analysis of Green Tissues from Plants

    PubMed Central

    Lassowskat, Ines; Naumann, Kai; Lee, Justin; Scheel, Dierk

    2013-01-01

    Phosphorylation is an important post-translational protein modification with regulatory roles in diverse cellular signaling pathways. Despite recent advances in mass spectrometry, the detection of phosphoproteins involved in signaling is still challenging, as protein phosphorylation is typically transient and/or occurs at low levels. In green plant tissues, the presence of highly abundant proteins, such as the subunits of the RuBisCO complex, further complicates phosphoprotein analysis. Here, we describe a simple, but powerful, method, which we named prefractionation-assisted phosphoprotein enrichment (PAPE), to increase the yield of phosphoproteins from Arabidopsis thaliana leaf material. The first step, a prefractionation via ammonium sulfate precipitation, not only depleted RuBisCO almost completely, but, serendipitously, also served as an efficient phosphoprotein enrichment step. When coupled with a subsequent metal oxide affinity chromatography (MOAC) step, the phosphoprotein content was highly enriched. The reproducibility and efficiency of phosphoprotein enrichment was verified by phospho-specific staining and, further, by mass spectrometry, where it could be shown that the final PAPE fraction contained a significant number of known and additionally novel (potential) phosphoproteins. Hence, this facile two-step procedure is a good prerequisite to probe the phosphoproteome and gain deeper insight into plant phosphorylation-based signaling events. PMID:28250405

  1. Large-scale phosphoproteome of human whole saliva using disulfide-thiol interchange covalent chromatography and mass spectrometry.

    PubMed

    Salih, Erdjan; Siqueira, Walter L; Helmerhorst, Eva J; Oppenheim, Frank G

    2010-12-01

    To date, only a handful of phosphoproteins with important biological functions have been identified and characterized in oral fluids, and these include some of the abundant protein constituents of saliva. Whole saliva (WS) samples were trypsin digested, followed by chemical derivatization using dithiothreitol (DTT) of the phospho-serine/threonine-containing peptides. The DTT-phosphopeptides were enriched by covalent disulfide-thiol interchange chromatography and analysis by nanoflow liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The specificity of DTT chemical derivatization was evaluated separately under different base-catalyzed conditions with NaOH and Ba(OH)(2), blocking cysteine residues by iodoacetamide and enzymatic O-deglycosylation prior to DTT reaction. Further analysis of WS samples that were subjected to either of these conditions provided supporting evidence for phosphoprotein identifications. The combined chemical strategies and mass spectrometric analyses identified 65 phosphoproteins in WS; of these, 28 were based on two or more peptide identification criteria with high confidence and 37 were based on a single phosphopeptide identification. Most of the identified proteins (∼80%) were previously unknown phosphoprotein components. This study represents the first large-scale documentation of phosphoproteins of WS. The origins and identity of WS phosphoproteome suggest significant implications for both basic science and the development of novel biomarkers/diagnostic tools for systemic and oral disease states.

  2. Dynamic Phosphoproteome Analysis of Seedling Leaves in Brachypodium distachyon L. Reveals Central Phosphorylated Proteins Involved in the Drought Stress Response

    PubMed Central

    Yuan, Lin-Lin; Zhang, Ming; Yan, Xing; Bian, Yan-Wei; Zhen, Shou-Min; Yan, Yue-Ming

    2016-01-01

    Drought stress is a major abiotic stress affecting plant growth and development. In this study, we performed the first dynamic phosphoproteome analysis of Brachypodium distachyon L. seedling leaves under drought stress for different times. A total of 4924 phosphopeptides, contained 6362 phosphosites belonging to 2748 phosphoproteins. Rigorous standards were imposed to screen 484 phosphorylation sites, representing 442 unique phosphoproteins. Comparative analyses revealed significant changes in phosphorylation levels at 0, 6, and 24 h under drought stress. The most phosphorylated proteins and the highest phosphorylation level occurred at 6 h. Venn analysis showed that the up-regulated phosphopeptides at 6 h were almost two-fold those at 24 h. Motif-X analysis identified the six motifs: [sP], [Rxxs], [LxRxxs], [sxD], [sF], and [TP], among which [LxRxxs] was also previously identified in B. distachyon. Results from molecular function and protein-protein interaction analyses suggested that phosphoproteins mainly participate in signal transduction, gene expression, drought response and defense, photosynthesis and energy metabolism, and material transmembrane transport. These phosphoproteins, which showed significant changes in phosphorylation levels, play important roles in signal transduction and material transmembrane transport in response to drought conditions. Our results provide new insights into the molecular mechanism of this plant’s abiotic stress response through phosphorylation modification. PMID:27748408

  3. Phosphoproteomic comparison of Pik3ca and Pten signalling identifies the nucleotidase NT5C as a novel AKT substrate

    PubMed Central

    Moniz, Larissa S.; Surinova, Silvia; Ghazaly, Essam; Velasco, Lorena Gonzalez; Haider, Syed; Rodríguez-Prados, Juan Carlos; Berenjeno, Inma M.; Chelala, Claude; Vanhaesebroeck, Bart

    2017-01-01

    To identify novel effectors and processes regulated by PI3K pathway activation, we performed an unbiased phosphoproteomic screen comparing two common events of PI3K deregulation in cancer: oncogenic Pik3ca mutation (Pik3caH1047R) and deletion of Pten. Using mouse embryonic fibroblast (MEF) models that generate inducible, low-level pathway activation as observed in cancer, we quantified 7566 unique phosphopeptides from 3279 proteins. A number of proteins were found to be differentially-regulated by Pik3caH1047R and Pten loss, suggesting unique roles for these two events in processes such as vesicular trafficking, DNA damage repair and RNA splicing. We also identified novel PI3K effectors that were commonly-regulated, including putative AKT substrates. Validation of one of these hits, confirmed NT5C (5′,3′-Nucleotidase, Cytosolic) as a novel AKT substrate, with an unexpected role in actin cytoskeleton regulation via an interaction with the ARP2/3 complex. This study has produced a comprehensive data resource and identified a new link between PI3K pathway activation and actin regulation. PMID:28059163

  4. Global Phosphoproteomic Analysis Reveals the Involvement of Phosphorylation in Aflatoxins Biosynthesis in the Pathogenic Fungus Aspergillus flavus

    PubMed Central

    Ren, Silin; Yang, Mingkun; Li, Yu; Zhang, Feng; Chen, Zhuo; Zhang, Jia; Yang, Guang; Yue, Yuewei; Li, Siting; Ge, Feng; Wang, Shihua

    2016-01-01

    Aspergillus flavus is a pathogenic fungus that produces toxic and carcinogenic aflatoxins and is the causative agent of aflatoxicosis. A growing body of evidence indicates that reversible phosphorylation plays important roles in regulating diverse functions in this pathogen. However, only a few phosphoproteins of this fungus have been identified, which hampers our understanding of the roles of phosphorylation in A. flavus. So we performed a global and site-specific phosphoproteomic analysis of A. flavus. A total of 598 high-confidence phosphorylation sites were identified in 283 phosphoproteins. The identified phosphoproteins were involved in various biological processes, including signal transduction and aflatoxins biosynthesis. Five identified phosphoproteins associated with MAPK signal transduction and aflatoxins biosynthesis were validated by immunoblotting using phospho-specific antibodies. Further functional studies revealed that phosphorylation of the MAP kinase kinase kinase Ste11 affected aflatoxins biosynthesis in A. flavus. Our data represent the results of the first global survey of protein phosphorylation in A. flavus and reveal previously unappreciated roles for phosphorylation in the regulation of aflatoxins production. The generated dataset can serve as an important resource for the functional analysis of protein phosphorylation in A. flavus and facilitate the elucidation of phosphorylated signaling networks in this pathogen. PMID:27667718

  5. Phosphoproteomic analysis of Methanohalophilus portucalensis FDF1T identified the role of protein phosphorylation in methanogenesis and osmoregulation

    PubMed Central

    Wu, Wan-Ling; Lai, Shu-Jung; Yang, Jhih-Tian; Chern, Jeffy; Liang, Suh-Yuen; Chou, Chi-Chi; Kuo, Chih-Horng; Lai, Mei-Chin; Wu, Shih-Hsiung

    2016-01-01

    Methanogens have gained much attention for their metabolic product, methane, which could be an energy substitute but also contributes to the greenhouse effect. One factor that controls methane emission, reversible protein phosphorylation, is a crucial signaling switch, and phosphoproteomics has become a powerful tool for large-scale surveying. Here, we conducted the first phosphorylation-mediated regulation study in halophilic Methanohalophilus portucalensis FDF1T, a model strain for studying stress response mechanisms in osmoadaptation. A shotgun approach and MS-based analysis identified 149 unique phosphoproteins. Among them, 26% participated in methanogenesis and osmolytes biosynthesis pathways. Of note, we uncovered that protein phosphorylation might be a crucial factor to modulate the pyrrolysine (Pyl) incorporation and Pyl-mediated methylotrophic methanogenesis. Furthermore, heterologous expression of glycine sarcosine N-methyltransferase (GSMT) mutant derivatives in the osmosensitive Escherichia coli MKH13 revealed that the nonphosphorylated T68A mutant resulted in increased salt tolerance. In contrast, mimic phosphorylated mutant T68D proved defective in both enzymatic activity and salinity tolerance for growth. Our study provides new insights into phosphorylation modification as a crucial role of both methanogenesis and osmoadaptation in methanoarchaea, promoting biogas production or reducing future methane emission in response to global warming and climate change. PMID:27357474

  6. Proteomic and phosphoproteomic analysis of renal cortex in a salt-load rat model of advanced kidney damage

    PubMed Central

    Jiang, Shaoling; He, Hanchang; Tan, Lishan; Wang, Liangliang; Su, Zhengxiu; Liu, Yufeng; Zhu, Hongguo; Zhang, Menghuan; Hou, Fan Fan; Li, Aiqing

    2016-01-01

    Salt plays an essential role in the progression of chronic kidney disease and hypertension. However, the mechanisms underlying pathogenesis of salt-induced kidney damage remain largely unknown. Here, Sprague-Dawley rats, that underwent 5/6 nephrectomy (5/6Nx, a model of advanced kidney damage) or sham operation, were treated for 2 weeks with a normal or high-salt diet. We employed aTiO2 enrichment, iTRAQ labeling and liquid-chromatography tandem mass spectrometry strategy for proteomic and phosphoproteomic profiling of the renal cortex. We found 318 proteins differentially expressed in 5/6Nx group relative to sham group, and 310 proteins significantly changed in response to salt load in 5/6Nx animals. Totally, 1810 unique phosphopeptides corresponding to 550 phosphoproteins were identified. We identified 113 upregulated and 84 downregulated phosphopeptides in 5/6Nx animals relative to sham animals. Salt load induced 78 upregulated and 91 downregulated phosphopeptides in 5/6Nx rats. The differentially expressed phospholproteins are important transporters, structural molecules, and receptors. Protein-protein interaction analysis revealed that the differentially phosphorylated proteins in 5/6Nx group, Polr2a, Srrm1, Gsta2 and Pxn were the most linked. Salt-induced differential phosphoproteins, Myh6, Lmna and Des were the most linked. Altered phosphorylation levels of lamin A and phospholamban were validated. This study will provide new insight into pathogenetic mechanisms of chronic kidney disease and salt sensitivity. PMID:27775022

  7. The Effect of a Prior Dissection Simulation on Middle School Students' Dissection Performance and Understanding of the Anatomy and Morphology of the Frog.

    ERIC Educational Resources Information Center

    Akpan, Joseph Paul; Andre, Thomas

    1999-01-01

    Reports on a study that examined the prior use of simulation on frog dissection in improving students' actual dissection performance and learning of frog anatomy and morphology. Finds that students who performed the simulation before dissection performed significantly better than students who performed dissection before using the simulation or…

  8. A Modified Dissection Method to Preserve Neck Structures

    ERIC Educational Resources Information Center

    Hankin, Mark H.; Stoller, Jeremy L.

    2009-01-01

    The neck is not only one of the more challenging anatomical regions to dissect but also has important application to clinical conditions, diseases, and procedures. In this study, we describe two simple modifications for dissection of the neck that (1) aid in the identification and preservation of the cutaneous branches of the cervical plexus and…

  9. Alternatives To Animal Dissection in School Science Classes. ERIC Digest.

    ERIC Educational Resources Information Center

    Haury, David L.

    Until recently, one of the most expected and accepted experiences among students in biology classrooms of the United States has been the dissection of vertebrate animals, from frogs and mice to cats and fetal pigs. However, resistance to animal dissection has grown during the past decade with concerns ranging from inhumane treatment of animals by…

  10. Local repair of distal thoracal aortic dissections (Locus minoris resistencia).

    PubMed

    Belov, Iu V; Komarov, R N; Stepanenko, A B; Gens, A P; Charchian, E R

    2007-01-01

    The paper presents the method of local repair of distal aortic dissections. Local aortic grafting for surgical correction of type B dissecting aortic aneurysms helped to decrease hospital mortality up to 15.4%, the rate of paraparesis and multiorgan failure - up to 11.5%.

  11. Outcomes of a Rotational Dissection System in Gross Anatomy

    ERIC Educational Resources Information Center

    Marshak, David W.; Oakes, Joanne; Hsieh, Pei-Hsuan; Chuang, Alice Z.; Cleary, Leonard J.

    2015-01-01

    At the University of Texas Houston Medical School, a rotational dissection system was introduced to improve coordination between the Gross Anatomy and the Introduction to Clinical Medicine (ICM) courses. Six students were assigned to each cadaver and divided into two teams. For each laboratory, one team was assigned to dissect and the other to…

  12. Dissection Videos Do Not Improve Anatomy Examination Scores

    ERIC Educational Resources Information Center

    Mahmud, Waqas; Hyder, Omar; Butt, Jamaal; Aftab, Arsalan

    2011-01-01

    In this quasi-experimental study, we describe the effect of showing dissection videos on first-year medical students' performance in terms of test scores during a gross anatomy course. We also surveyed students' perception regarding the showing of dissection videos. Two hundred eighty-seven first-year medical students at Rawalpindi Medical College…

  13. Student Attitudes toward Cadaveric Dissection at a UK Medical School

    ERIC Educational Resources Information Center

    Quince, Thelma A.; Barclay, Stephen I. G.; Spear, Michelle; Parker, Richard A.; Wood, Diana F.

    2011-01-01

    A more humanistic approach toward dissection has emerged. However, student attitudes toward this approach are unknown and the influences on such attitudes are little understood. One hundred and fifty-six first-year medical students participated in a study examining firstly, attitudes toward the process of dissection and the personhood of the…

  14. Recurrent spontaneous coronary artery dissection: acute management and literature review

    PubMed Central

    Dana, Ali

    2012-01-01

    Spontaneous coronary artery dissection is a rare cause of acute presentations to the catheter laboratory. Often, the angiographic findings are subtle and may be mistaken for a plaque rupture. We descibe a case where repeat presentation revealed the diagnosis of recurrent spontaneous coronary artery dissection. PMID:24062889

  15. Evaluating Multiplexed Quantitative Phosphopeptide Analysis on a Hybrid Quadrupole Mass Filter/Linear Ion Trap/Orbitrap Mass Spectrometer

    PubMed Central

    2015-01-01

    As a driver for many biological processes, phosphorylation remains an area of intense research interest. Advances in multiplexed quantitation utilizing isobaric tags (e.g., TMT and iTRAQ) have the potential to create a new paradigm in quantitative proteomics. New instrumentation and software are propelling these multiplexed workflows forward, which results in more accurate, sensitive, and reproducible quantitation across tens of thousands of phosphopeptides. This study assesses the performance of multiplexed quantitative phosphoproteomics on the Orbitrap Fusion mass spectrometer. Utilizing a two-phosphoproteome model of precursor ion interference, we assessed the accuracy of phosphopeptide quantitation across a variety of experimental approaches. These methods included the use of synchronous precursor selection (SPS) to enhance TMT reporter ion intensity and accuracy. We found that (i) ratio distortion remained a problem for phosphopeptide analysis in multiplexed quantitative workflows, (ii) ratio distortion can be overcome by the use of an SPS-MS3 scan, (iii) interfering ions generally possessed a different charge state than the target precursor, and (iv) selecting only the phosphate neutral loss peak (single notch) for the MS3 scan still provided accurate ratio measurements. Remarkably, these data suggest that the underlying cause of interference may not be due to coeluting and cofragmented peptides but instead from consistent, low level background fragmentation. Finally, as a proof-of-concept 10-plex experiment, we compared phosphopeptide levels from five murine brains to five livers. In total, the SPS-MS3 method quantified 38 247 phosphopeptides, corresponding to 11 000 phosphorylation sites. With 10 measurements recorded for each phosphopeptide, this equates to more than 628 000 binary comparisons collected in less than 48 h. PMID:25521595

  16. Traditional versus Computer-Based Dissections in Enhancing Learning in a Tertiary Setting: A Student Perspective.

    ERIC Educational Resources Information Center

    Franklin, Sue; Peat, Mary; Lewis, Alison

    2002-01-01

    Describes a study that investigates both the use and usefulness of laboratory dissections and computer-based dissections in a tertiary, first-year human biology course. Explores attitudes toward dissection. (DDR)

  17. CrossFit-related cervical internal carotid artery dissection.

    PubMed

    Lu, Albert; Shen, Peter; Lee, Paul; Dahlin, Brian; Waldau, Ben; Nidecker, Anna E; Nundkumar, Anoop; Bobinski, Matthew

    2015-08-01

    CrossFit is a high-intensity strength and conditioning program that has gained popularity over the past decade. Potential injuries associated with CrossFit training have been suggested in past reports. We report three cases of cervical carotid dissection that are associated with CrossFit workouts. Patient 1 suffered a distal cervical internal carotid artery (ICA) dissection near the skull base and a small infarct in Wernicke's area. He was placed on anticoagulation and on follow-up has near complete recovery. Patient 2 suffered a proximal cervical ICA dissection that led to arterial occlusion and recurrent middle cerebral artery territory infarcts and significant neurological sequelae. Patient 3 had a skull base ICA dissection that led to a partial Horner's syndrome but no cerebral infarct. While direct causality cannot be proven, intense CrossFit workouts may have led to the ICA dissections in these patients.

  18. Impact of bilateral neck dissection on recovery following supraglottic laryngectomy.

    PubMed

    Weber, P C; Johnson, J T; Myers, E N

    1993-01-01

    Previously reported data from our institution has led us to perform bilateral neck dissections for therapeutic as well as staging advantages for horizontal supraglottic laryngectomies. Concern over the possibility of increased morbidity associated with simultaneous bilateral neck dissection prompted this retrospective review of patients with supraglottic laryngectomy who were treated with either unilateral (46 patients) or bilateral (23 patients) neck dissection. No significant differences were found in morbidity when patients were evaluated for transfusion rate, cervical wound drainage, need for tracheotomy, oral diet, or duration of hospitalization. Significant differences were noted in surgical operating time, eg, it took 100 minutes longer to perform bilateral dissections, and slight increases were noted in estimated blood loss and fluids given intravenously. No significant differences were noted in the percentage or type of postoperative complications. It seems that bilateral neck dissection in conjunction with supraglottic laryngectomy does not increase postoperative surgical morbidity and may actually avoid complications associated with postoperative radiation therapy in patients with supraglottic laryngectomy.

  19. Lymph node dissection for gastric cancer: a critical review

    PubMed Central

    Batista, Thales Paulo; Martins, Mário Rino

    2012-01-01

    Gastric cancer is one of the most common neoplasms and an important cause of cancer-related death worldwide. Efforts to reduce its high mortality rates are currently focused on multidisciplinary management. However, surgery remains a cornerstone in the management of patients with resectable disease. There is still some controversy as to the extent of lymph node dissection for potentially curable stomach cancer. Surgeons in eastern countries favor more extensive lymph node dissection, whereas those in the West favor less extensive dissection. Thus, extent of lymph node dissection remains one of the most hotly discussed aspects of gastric surgery, particularly because most stomach cancers are now often comprehensively treated by adding some perioperative chemotherapy or chemo-radiation. We provide a critical review of lymph nodes dissection for gastric cancer with a particular focus on its benefits in a multimodal approach. PMID:25992202

  20. Controlled ultrasonic micro-dissection of thin tissue sections.

    PubMed

    Ru, Changhai; Liu, Jun; Pang, Ming; Sun, Yu

    2014-08-01

    In order to obtain sufficient quantities of pure populations of cells or a single cell from surrounding tissue for analytical investigation, we have developed an ultrasonic microdissection system. The system utilizes a vision-based method for detecting the contact between the microdissection needle tip and a target surface. A multilayer stack piezoelectric actuator is employed to generate ultrasonic vibrations for histological isolation. Automated micro-dissection is also realized using visual feedback and vision-based control. Experimental results on tumor tissue sections show that the system has a high dissection accuracy and efficiency and is able to realize dissecting arbitrary shapes in specified locations on a tissue sample. Furthermore, effects in variations of vibration amplitude and frequency of ultrasonic micro-dissection as well as needle insertion depths on micro-dissection accuracy and speed were evaluated.

  1. Dissecting Japan's Dengue Outbreak in 2014

    PubMed Central

    Quam, Mikkel B.; Sessions, October; Kamaraj, Uma Sangumathi; Rocklöv, Joacim; Wilder-Smith, Annelies

    2016-01-01

    Despite Japan's temperate climate, a dengue outbreak occurred in Tokyo for the first time in over 70 years in 2014. We dissected this dengue outbreak based on phylogenetic analysis, travel interconnectivity, and environmental drivers for dengue epidemics. Comparing the available dengue virus 1 (DENV1) E gene sequence from this outbreak with 3,282 unique DENV1 sequences in National Center for Biotechnology Information suggested that the DENV might have been imported from China, Indonesia, Singapore, or Vietnam. With travelers arriving into Japan, Guangzhou (China) may have been the source of DENV introduction, given that Guangzhou also reported a large-scale dengue outbreak in 2014. Coinciding with the 2014 outbreak, Tokyo's climate conditions permitted the amplification of Aedes vectors and the annual peak of vectorial capacity. Given suitable vectors and climate conditions in addition to increasing interconnectivity with endemic areas of Asia, Tokyo's 2014 outbreak did not come as a surprise and may foretell more to come. PMID:26711518

  2. Recent traction methods for endoscopic submucosal dissection

    PubMed Central

    Tsuji, Kunihiro; Yoshida, Naohiro; Nakanishi, Hiroyoshi; Takemura, Kenichi; Yamada, Shinya; Doyama, Hisashi

    2016-01-01

    Endoscopic mucosal resection (EMR) is problematic with regard to en bloc and curable resection rates. Advancements in endoscopic techniques have enabled novel endoscopic approaches such as endoscopic submucosal dissection (ESD), which has overcome some EMR problems, and has become the standard treatment for gastrointestinal tumors. However, ESD is technically difficult. Procedure time is longer and complications such as intraoperative perforation and bleeding occur more frequently than in EMR. Recently various traction methods have been introduced to facilitate ESD procedures, such as clip with line, external forceps, clip and snare, internal traction, double scope, and magnetic anchor. Each method must be used appropriately according to the anatomical characteristics. In this review we discuss recently proposed traction methods for ESD based on the characteristics of various anatomical sites. PMID:27468186

  3. Chemical Genetics in Dissecting Membrane Glycerolipid Functions.

    PubMed

    Chevalier, Florian; Carrera, Laura Cuyàs; Nussaume, Laurent; Maréchal, Eric

    2016-01-01

    Chemical genetics has emerged as a powerful approach to dissect biological processes, based on the utilization of small molecules disturbing the function of specific target proteins. By analogy with classical genetics, 'reverse chemical genetics' refers to the utilization of drugs acting on a known target, enabling its functional characterization at the levels of the cells, tissues and organisms. Likewise, 'direct chemical genetics' refers to the utilization of a drug of unknown mode of action, but triggering a phenotype of interest. In that case, one has to identify the target(s) possibly blocked (or possibly activated) by the small molecule. This chapter illustrates both approaches, like the analysis of the elongation of fatty acids, the biosynthesis of galactoglycerolipids or the catabolism of phosphoglycerolipids by reverse chemical genetics or the study of the membrane glycerolipid remodeling triggered upon phosphate starvation, by direct chemical genetics.

  4. Contemporary Review on Spontaneous Coronary Artery Dissection.

    PubMed

    Saw, Jacqueline; Mancini, G B John; Humphries, Karin H

    2016-07-19

    Spontaneous coronary artery dissection (SCAD) is gaining recognition as an important cause of myocardial infarction, especially in young women. There has been a surge in the diagnosis of SCAD in recent years, presumably due to an increased use of coronary angiography, and the clinical availability and application of high-resolution intracoronary imaging. The improved recognition and diagnosis, together with increased publications and attention through social media, have considerably raised awareness of this condition, which was once believed to be very rare. Recent publications of moderate to large contemporary case series have helped elucidate the early natural history, presenting characteristics (clinical and angiographic), underlying etiology, management, and cardiovascular outcomes with this condition, thus providing observations and important clinical insights of value to clinicians managing this challenging and perplexing patient cohort. The aim of our review is to provide a comprehensive contemporary update of SCAD to aid health care professionals in managing these patients in both the acute and chronic settings.

  5. Nanoscale scraping and dissection of collagen fibrils.

    PubMed

    Wenger, M P E; Horton, M A; Mesquida, P

    2008-09-24

    The main function of collagen is mechanical, hence there is a fundamental scientific interest in experimentally investigating the mechanical and structural properties of collagen fibrils on the nanometre scale. Here, we present a novel atomic force microscopy (AFM) based scraping technique that can dissect the outer layer of a biological specimen. Applied to individual collagen fibrils, the technique was successfully used to expose the fibril core and reveal the presence of a D-banding-like structure. AFM nanoindentation measurements of fibril shell and core indicated no significant differences in mechanical properties such as stiffness (reduced modulus), hardness, adhesion and adhesion work. This suggests that collagen fibrils are mechanically homogeneous structures. The scraping technique can be applied to other biological specimens, as demonstrated on the example of bacteria.

  6. Influence of a Dissection Video Clip on Anxiety, Affect, and Self-Efficacy in Educational Dissection: A Treatment Study

    PubMed Central

    Randler, Christoph; Demirhan, Eda; Wüst-Ackermann, Peter; Desch, Inga H.

    2016-01-01

    In science education, dissections of animals are an integral part of teaching, but they often evoke negative emotions. We aimed at reducing negative emotions (anxiety, negative affect [NA]) and increasing positive affect (PA) and self-efficacy by an experimental intervention using a predissection video to instruct students about fish dissection. We compared this treatment with another group that watched a life history video about the fish. The participants were 135 students studying to become biology teachers. Seventy received the treatment with the dissection video, and 65 viewed the life history video. We applied a pre/posttest treatment-comparison design and used the Positive and Negative Affect Schedule (PANAS), the State–Trait–Anxiety Inventory for State (STAI-S), and a self-efficacy measure three times: before the lesson (pretest), after the film treatment (posttest 1), and after the dissection (posttest 2). The dissection film group scored higher in PA, NA, and state anxiety (STAI-S) after the dissection video treatment and higher in self-efficacy after the dissection. The life history group showed no differences between the pretest and posttest 1. The dissection film has clear benefits—increasing PA and self-efficacy—that come at the cost of higher NA and higher STAI-S. PMID:27290738

  7. Influence of a Dissection Video Clip on Anxiety, Affect, and Self-Efficacy in Educational Dissection: A Treatment Study

    ERIC Educational Resources Information Center

    Randler, Christoph; Demirhan, Eda; Wüst-Ackermann, Peter; Desch, Inga H.

    2016-01-01

    In science education, dissections of animals are an integral part of teaching, but they often evoke negative emotions. We aimed at reducing negative emotions (anxiety, negative affect [NA]) and increasing positive affect (PA) and self-efficacy by an experimental intervention using a predissection video to instruct students about fish dissection.…

  8. The Effects of Computer Animated Dissection versus Preserved Animal Dissection on the Student Achievement in a High School Biology Class.

    ERIC Educational Resources Information Center

    Kariuki, Patrick; Paulson, Ronda

    The purpose of this study was to examine the effectiveness of computer-animated dissection techniques versus the effectiveness of traditional dissection techniques as related to student achievement. The sample used was 104 general biology students from a small, rural high school in Northeast Tennessee. Random selection was used to separate the…

  9. Influence of a Dissection Video Clip on Anxiety, Affect, and Self-Efficacy in Educational Dissection: A Treatment Study.

    PubMed

    Randler, Christoph; Demirhan, Eda; Wüst-Ackermann, Peter; Desch, Inga H

    2016-01-01

    In science education, dissections of animals are an integral part of teaching, but they often evoke negative emotions. We aimed at reducing negative emotions (anxiety, negative affect [NA]) and increasing positive affect (PA) and self-efficacy by an experimental intervention using a predissection video to instruct students about fish dissection. We compared this treatment with another group that watched a life history video about the fish. The participants were 135 students studying to become biology teachers. Seventy received the treatment with the dissection video, and 65 viewed the life history video. We applied a pre/posttest treatment-comparison design and used the Positive and Negative Affect Schedule (PANAS), the State-Trait-Anxiety Inventory for State (STAI-S), and a self-efficacy measure three times: before the lesson (pretest), after the film treatment (posttest 1), and after the dissection (posttest 2). The dissection film group scored higher in PA, NA, and state anxiety (STAI-S) after the dissection video treatment and higher in self-efficacy after the dissection. The life history group showed no differences between the pretest and posttest 1. The dissection film has clear benefits - increasing PA and self-efficacy - that come at the cost of higher NA and higher STAI-S.

  10. Phosphoproteomic Analysis Reveals a Novel Mechanism of CaMKIIα Regulation Inversely Induced by Cocaine Memory Extinction versus Reconsolidation

    PubMed Central

    Rich, Matthew T.; Abbott, Thomas B.; Chung, Lisa; Gulcicek, Erol E.; Stone, Kathryn L.; Colangelo, Christopher M.; Lam, TuKiet T.; Nairn, Angus C.; Taylor, Jane R.

    2016-01-01

    Successful addiction treatment depends on maintaining long-term abstinence, making relapse prevention an essential therapeutic goal. However, exposure to environmental cues associated with drug use often thwarts abstinence efforts by triggering drug using memories that drive craving and relapse. We sought to develop a dual approach for weakening cocaine memories through phosphoproteomic identification of targets regulated in opposite directions by memory extinction compared with reconsolidation in male Sprague-Dawley rats that had been trained to self-administer cocaine paired with an audiovisual cue. We discovered a novel, inversely regulated, memory-dependent phosphorylation event on calcium-calmodulin-dependent kinase II α (CaMKIIα) at serine (S)331. Correspondingly, extinction-associated S331 phosphorylation inhibited CaMKIIα activity. Intra-basolateral amygdala inhibition of CaMKII promoted memory extinction and disrupted reconsolidation, leading to a reduction in subsequent cue-induced reinstatement. CaMKII inhibition had no effect if the memory was neither retrieved nor extinguished. Therefore, inhibition of CaMKII represents a novel mechanism for memory-based addiction treatment that leverages both extinction enhancement and reconsolidation disruption to reduce relapse-like behavior. SIGNIFICANCE STATEMENT Preventing relapse to drug use is an important goal for the successful treatment of addictive disorders. Relapse-prevention therapies attempt to interfere with drug-associated memories, but are often hindered by unintentional memory strengthening. In this study, we identify phosphorylation events that are bidirectionally regulated by the reconsolidation versus extinction of a cocaine-associated memory, including a novel site on CaMKIIα. Additionally, using a rodent model of addiction, we show that CaMKII inhibition in the amygdala can reduce relapse-like behavior. Together, our data supports the existence of mechanisms that can be used to enhance

  11. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication

    PubMed Central

    Avey, Denis; Tepper, Sarah; Li, Wenwei; Turpin, Zachary; Zhu, Fanxiu

    2015-01-01

    Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling) during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5’ UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45 manipulates

  12. The Phosphoproteome of a Chlamydomonas reinhardtii Eyespot Fraction Includes Key Proteins of the Light Signaling Pathway1[W

    PubMed Central

    Wagner, Volker; Ullmann, Katharina; Mollwo, Anne; Kaminski, Marc; Mittag, Maria; Kreimer, Georg

    2008-01-01

    Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. In a recent proteomic approach, we identified 202 proteins from a fraction enriched in eyespot apparatuses of Chlamydomonas reinhardtii. Among these proteins, five protein kinases and two protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction, we analyzed its phosphoproteome. The solubilized proteins of the eyespot fraction were treated with the endopeptidases LysC and trypsin prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as neutral-loss-triggered MS/MS/MS spectra. We were able to identify 68 different phosphopeptides along with 52 precise in vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a Ca2+-binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively. Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly light-gated ion channels relevant for the photoresponses of C. reinhardtii. PMID:18065559

  13. Traumatic carotid artery dissection caused by bungee jumping.

    PubMed

    Zhou, Wei; Huynh, Tam T; Kougias, Panagiotis; El Sayed, Hosam F; Lin, Peter H

    2007-11-01

    Bungee jumping is a popular recreational activity in which participant experiences transient freefall while connected to a bungee cord. The rapid freefall and the resultant rebound force created by the bungee cord can result in a variety of bodily injuries. We report herein a case of traumatic carotid artery dissection caused by bungee jumping. The symptoms related to carotid artery dissection were successfully treated with anticoagulation. The etiology of carotid dissection related to bungee jumping is discussed. Physicians should be cognizant of this potential injury due to the force created by the freefall and rebound motion associated in this recreational sport.

  14. Thoraco-abdominal Aorta Dissection: Look Again Before You Leap.

    PubMed

    Zeina, Abdel-Rauf; Trachtengerts, Victoria; Abadi, Sobhi; Jarchowsky, Jacob; Soimu, Uri; Nachtigal, Alicia

    2009-01-01

    Aortic dissection is a life-threatening condition that might require immediate assessment and therapy. We present the case of a 71-year-old woman with essential hypertension complaining about low back pain; unenhanced thoracic-lumbar spine computed tomography examination (CT) revealed a hyperdense thin line across the aorta with an appearance of "double aortic lumen". Enhanced CT scan confirmed the diagnosis of type B aortic dissection. Radiologists should be familiar with this finding that could be considered a new radiological sign of aortic dissection on unenhanced CT examination.

  15. A Type A Aortic Dissection Mimicking an Acute Myocardial Infarction

    PubMed Central

    D’Aloia, Antonio; Vizzardi, Enrico; Bugatti, Silvia; Magatelli, Marco; Bonadei, Ivano; Rovetta, Riccardo; Quinzani, Filippo; Curnis, Antonio; Cas, Livio Dei

    2012-01-01

    We describe a case of a 54 years old man in whom an initial diagnosis of acute coronary syndrome (ACS) revealed to be finally an acute aortic dissection. This case report stresses the importance to maintain a high grade of suspicion of aortic dissection as a possible alternative in presence of eletrocardiographic myocardial ischemic signs. In many medical centers where thrombolitic therapy, antiplatelets receptor blockers, heparin or percutaneous coronary angioplasty is the first line therapy for ACS the outcome may be catastrophic in situation such as aortic dissection.

  16. Neck dissection with cervical sensory preservation in thyroid cancer.

    PubMed

    Xue, Shuai; Wang, Peisong; Chen, Guang

    2013-11-01

    Thyroid cancer is the most common endocrine malignancy. Recently, controversy has focused on the management of lymph node metastases, which represent approximately 90% of disease recurrences and may require considerable time, effort, and resources to diagnose and treat. Neck dissections play an essential role in the management of head and neck cancer. A modified radical neck dissection (MND) refers to resection of the lymph nodes in levels II through V and often including the central nodes in level VI. When performing modified neck dissection, we recommend to protect more reserved cervical plexus. The purpose is to better protect patient's neck skin feeling.

  17. Pediatric traumatic carotid, vertebral and cerebral artery dissections: a review.

    PubMed

    Mortazavi, Martin M; Verma, Ketan; Tubbs, R Shane; Harrigan, Mark

    2011-12-01

    Traumatic cerebral dissections are rare but potentially dangerous conditions that through improved diagnostics have recently gained increased interest. However, there is still a significant lack of knowledge on the natural history, as well as on the best treatment options. Most of the literature on this topic consists of case reports and retrospective studies with no prospective randomized controlled studies. In our review, we highlight the fact that there is no level 1 evidence for the natural history of cerebral dissections or for the best treatment. We present 26 case studies derived from 70 pediatric patients affected by dissections, occlusions, and pseudoaneurysms.

  18. Comparative phosphoproteomic analysis of mammalian glomeruli reveals conserved podocin C-terminal phosphorylation as a determinant of slit diaphragm complex architecture.

    PubMed

    Rinschen, Markus M; Pahmeyer, Caroline; Pisitkun, Trairak; Schnell, Nicole; Wu, Xiongwu; Maaß, Martina; Bartram, Malte P; Lamkemeyer, Tobias; Schermer, Bernhard; Benzing, Thomas; Brinkkoetter, Paul T

    2015-04-01

    Glomerular biology is dependent on tightly controlled signal transduction networks that control phosphorylation of signaling proteins such as cytoskeletal regulators or slit diaphragm proteins of kidney podocytes. Cross-species comparison of phosphorylation events is a powerful mean to functionally prioritize and identify physiologically meaningful phosphorylation sites. Here, we present the result of phosphoproteomic analyses of cow and rat glomeruli to allow cross-species comparisons. We discovered several phosphorylation sites with potentially high biological relevance, e.g. tyrosine phosphorylation of the cytoskeletal regulator synaptopodin and the slit diaphragm protein neph-1 (Kirrel). Moreover, cross-species comparisons revealed conserved phosphorylation of the slit diaphragm protein nephrin on an acidic cluster at the intracellular terminus and conserved podocin phosphorylation on the very carboxyl terminus of the protein. We studied a highly conserved podocin phosphorylation site in greater detail and show that phosphorylation regulates affinity of the interaction with nephrin and CD2AP. Taken together, these results suggest that species comparisons of phosphoproteomic data may reveal regulatory principles in glomerular biology. All MS data have been deposited in the ProteomeXchange with identifier PXD001005 (http://proteomecentral.proteomexchange.org/dataset/PXD001005).

  19. Optogenetic dissection of medial prefrontal cortex circuitry

    PubMed Central

    Riga, Danai; Matos, Mariana R.; Glas, Annet; Smit, August B.; Spijker, Sabine; Van den Oever, Michel C.

    2014-01-01

    The medial prefrontal cortex (mPFC) is critically involved in numerous cognitive functions, including attention, inhibitory control, habit formation, working memory and long-term memory. Moreover, through its dense interconnectivity with subcortical regions (e.g., thalamus, striatum, amygdala and hippocampus), the mPFC is thought to exert top-down executive control over the processing of aversive and appetitive stimuli. Because the mPFC has been implicated in the processing of a wide range of cognitive and emotional stimuli, it is thought to function as a central hub in the brain circuitry mediating symptoms of psychiatric disorders. New optogenetics technology enables anatomical and functional dissection of mPFC circuitry with unprecedented spatial and temporal resolution. This provides important novel insights in the contribution of specific neuronal subpopulations and their connectivity to mPFC function in health and disease states. In this review, we present the current knowledge obtained with optogenetic methods concerning mPFC function and dysfunction and integrate this with findings from traditional intervention approaches used to investigate the mPFC circuitry in animal models of cognitive processing and psychiatric disorders. PMID:25538574

  20. Esophageal Stricture Prevention after Endoscopic Submucosal Dissection

    PubMed Central

    Jain, Deepanshu; Singhal, Shashideep

    2016-01-01

    Advances in diagnostic modalities and improvement in surveillance programs for Barrett esophagus has resulted in an increase in the incidence of superficial esophageal cancers (SECs). SEC, due to their limited metastatic potential, are amenable to non-invasive treatment modalities. Endoscopic ultrasound, endoscopic mucosal resection, and endoscopic submucosal dissection (ESD) are some of the new modalities that gastroenterologists have used over the last decade to diagnose and treat SEC. However, esophageal stricture (ES) is a very common complication and a major cause of morbidity post-ESD. In the past few years, there has been a tremendous effort to reduce the incidence of ES among patients undergoing ESD. Steroids have shown the most consistent results over time with minimal complications although the preferred mode of delivery is debatable, with both systemic and local therapy having pros and cons for specific subgroups of patients. Newer modalities such as esophageal stents, autologous cell sheet transplantation, polyglycolic acid, and tranilast have shown promising results but the depth of experience with these methods is still limited. We have summarized case reports, prospective single center studies, and randomized controlled trials describing the various methods intended to reduce the incidence of ES after ESD. Indications, techniques, outcomes, limitations, and reported complications are discussed. PMID:26949124

  1. Endovascular treatment of acute type B dissection complicating aortic coarctation.

    PubMed

    Kassaian, Seyed Ebrahim; Abbasi, Kyomars; Mousavi, Mehdi; Sahebjam, Mohammad

    2013-01-01

    Surgical treatment poses a high risk to patients with concomitant aortic coarctation and dissection, and an interventional approach could be an alternative. We describe the case of a 52-year-old man with a long history of untreated hypertension and aortic coarctation who emergently presented at our institution with an acute Stanford type B dissection. The patient's elevated serum creatinine level, perfusion deficit in the right lower limb, and hypertension did not respond to medical therapy, and he did not consent to surgery. By endovascular means, we used a self-expandable stent-graft to cover the entry point of the dissection; then, we deployed a balloon-expandable bare-metal stent to correct residual stenosis. To our knowledge, this is the first report of the endovascular treatment of aortic coarctation complicated by type B dissection.

  2. D-dimer is elevated in acute aortic dissection.

    PubMed

    Martin, Thomas; Shariq, Sohail

    2010-08-31

    This case illustrates that d-dimer is elevated in patients with acute aortic dissection. A 49-year-old woman presented with central, crushing chest pain exacerbated on inspiration. The chest pain was associated with right-leg numbness and pain, although peripheral pulses and blood pressures were normal. Routine bloods demonstrated an elevated d-dimer with a normal ECG and chest x-ray radiograph. A differential diagnosis of pulmonary embolism and acute aortic dissection was made. CT-angiogram showed type B aortic dissection. This case report highlights the mounting evidence that d-dimer is elevated in practically all incidents of aortic dissection and could be useful as a negative predictive marker.

  3. Complicated Postpartum Type B Aortic Dissection and Endovascular Repair

    PubMed Central

    Rosenberger, Laura H.; Adams, Joshua D.; Kern, John A.; Tracci, Margaret C.; Angle, J. Fritz; Cherry, Kenneth J.

    2012-01-01

    BACKGROUND Fifty percent of aortic dissections in women younger than 40 years occur in association with pregnancy. Of these, half of type B dissections occur in the postpartum period. CASE A 30-year-old woman was status post spontaneous vaginal delivery at 30 weeks of gestation for fetal death, complicated by an eclamptic seizure. On post-partum day 4, she suffered an acute, complicated type B aortic dissection treated with endovascular stent graft placement. CONCLUSION Endovascular repair may be an attractive option for the treatment of complicated type B aortic dissections in pregnancy and the peripartum period, with reduced maternal and fetal mortality. This may allow the fetus to remain in situ and avoid the risks of surgery and possible cardiopulmonary bypass, with little radiation risk to the fetus. PMID:22270446

  4. SMAD2 Mutations Are Associated with Arterial Aneurysms and Dissections.

    PubMed

    Micha, Dimitra; Guo, Dong-Chuan; Hilhorst-Hofstee, Yvonne; van Kooten, Fop; Atmaja, Dian; Overwater, Eline; Cayami, Ferdy K; Regalado, Ellen S; van Uffelen, René; Venselaar, Hanka; Faradz, Sultana M H; Vriend, Gerrit; Weiss, Marjan M; Sistermans, Erik A; Maugeri, Alessandra; Milewicz, Dianna M; Pals, Gerard; van Dijk, Fleur S

    2015-12-01

    We report three families with arterial aneurysms and dissections in which variants predicted to be pathogenic were identified in SMAD2. Moreover, one variant occurred de novo in a proband with unaffected parents. SMAD2 is a strong candidate gene for arterial aneurysms and dissections given its role in the TGF-β signaling pathway. Furthermore, although SMAD2 and SMAD3 probably have functionally distinct roles in cell signaling, they are structurally very similar. Our findings indicate that SMAD2 mutations are associated with arterial aneurysms and dissections and are in accordance with the observation that patients with pathogenic variants in genes encoding proteins involved in the TGF-β signaling pathway exhibit arterial aneurysms and dissections as key features.

  5. Dissection of the right coronary artery following blunt cardiac injury

    PubMed Central

    Vogiatzis, I; Dapcevic, I

    2015-01-01

    Background Coronary artery dissection is a rare complication of blunt thoracic trauma which can become rapidly lethal necessitating prompt diagnosis and treatment. Most reported cases of coronary artery injury, including dissection, involve the left anterior descending coronary artery, given its anatomical location in relation to the impact. Description of case A 72-year-old male, who was involved in a vehicular accident, sustained blunt thoracic trauma which resulted in isolated right coronary artery dissection and acute myocardial infarction. The culprit lesion was found in coronary angiography in the proximal right coronary artery and was successfully repaired with percutaneous coronary intervention and one drug-eluting stent placement. Conclusion Traumatic dissection of coronary arteries must be suspected in blunt thoracic trauma. It can be treated with interventional management and results in a fairly good prognosis. Hippokratia 2015; 19 (3): 278-280. PMID:27418793

  6. Coronary Artery Dissection: Not Just a Heart Attack

    MedlinePlus

    ... Aneurysm More Coronary Artery Dissection: Not Just a Heart Attack Updated:Oct 4,2016 Sometimes a heart attack ... Disease Go Red For Women Types of aneurysms Heart Attack • Home • About Heart Attacks Acute Coronary Syndrome (ACS) ...

  7. Quantitative research.

    PubMed

    Watson, Roger

    2015-04-01

    This article describes the basic tenets of quantitative research. The concepts of dependent and independent variables are addressed and the concept of measurement and its associated issues, such as error, reliability and validity, are explored. Experiments and surveys – the principal research designs in quantitative research – are described and key features explained. The importance of the double-blind randomised controlled trial is emphasised, alongside the importance of longitudinal surveys, as opposed to cross-sectional surveys. Essential features of data storage are covered, with an emphasis on safe, anonymous storage. Finally, the article explores the analysis of quantitative data, considering what may be analysed and the main uses of statistics in analysis.

  8. [Aortic dissection and pregnancy. Apropos of a case].

    PubMed

    Helms, E; Uguen, T; Amaranto, P; Carton, M J; Ducreux, J C; Tempelhoff, C

    1995-03-01

    Aortic dissection is a serious disease which rarely affects young women. In this context, it occurs in nearly one out of two cases during pregnancy, usually during the third term. The authors report acute dissection of the ascending aorta (de Bakey type 2) during pregnancy for which rapid cardiothoracic surgical management as a semi-emergency resulted in a favourable outcome for mother and child.

  9. Spontaneous Bilateral Vertebral Artery Dissection During a Basketball Game

    PubMed Central

    Mas Rodriguez, Manuel F.; Berrios, Rafael Arias; Ramos, Edwardo

    2016-01-01

    Spontaneous vertebral artery dissection accounts for 2% of all ischemic strokes and can occur as a consequence of sports events. We present an unusual case of spontaneous bilateral vertebral artery dissection in a 30-year-old male patient during a basketball game. He developed severe dysphagia, right hemiparesis, and balance dysfunction. We also present a review of the pathology, diagnosis, symptomatology, treatment, prognosis, and occurrence of this entity in sports. PMID:26733592

  10. Dissection of a non-bifurcating cervical carotid artery.

    PubMed

    Nas, Omer Fatih; Karakullukcuoglu, Zeynel; Hakyemez, Bahattin; Erdogan, Cuneyt

    2016-06-01

    A non-bifurcating cervical carotid artery is a rare anomaly in the population. Radiologic diagnosis of pathologies seen together with this anomaly can be challenging. Despite not being diagnostic all the time, digital subtraction angiography is accepted as the gold standard method for the diagnosis of dissection. We present a case of a non-bifurcating cervical carotid artery and concomitant dissection, which presented to the hospital with trauma and ischemic findings.

  11. Silent aortic dissection presenting as transient locked-in syndrome.

    PubMed

    Nadour, Wadih; Goldwasser, Brian; Biederman, Robert W; Taffe, Kevin

    2008-01-01

    Acute aortic dissection is a medical emergency. Without prompt recognition and treatment, the mortality rate is high. An atypical presentation makes timely diagnosis difficult, especially if the patient is experiencing no characteristic pain. Many patients with aortic dissection are reported to have presented with various neurologic manifestations, but none with only a presentation of transient locked-in syndrome.Herein, we report a case of completely painless aortic dissection in a woman who presented with a transient episode of anarthria, quadriplegia, and preserved consciousness. On physical examination, she had a 40-point difference in blood pressure between her left and right arms, and a loud diastolic murmur. The diagnosis of acute aortic dissection was reached via a combination of radiography, computed tomography, echocardiography, and a high index of clinical suspicion. The patient underwent emergency surgery and ultimately experienced a successful outcome.To our knowledge, this is the 1st report of aortic dissection that presented solely as locked-in syndrome. We suggest that silent aortic dissection be added to the differential diagnosis for transient locked-in syndrome.

  12. Epidemiology, pathophysiology, diagnosis, and management of intracranial artery dissection.

    PubMed

    Debette, Stéphanie; Compter, Annette; Labeyrie, Marc-Antoine; Uyttenboogaart, Maarten; Metso, Tina M; Majersik, Jennifer J; Goeggel-Simonetti, Barbara; Engelter, Stefan T; Pezzini, Alessandro; Bijlenga, Philippe; Southerland, Andrew M; Naggara, Olivier; Béjot, Yannick; Cole, John W; Ducros, Anne; Giacalone, Giacomo; Schilling, Sabrina; Reiner, Peggy; Sarikaya, Hakan; Welleweerd, Janna C; Kappelle, L Jaap; de Borst, Gert Jan; Bonati, Leo H; Jung, Simon; Thijs, Vincent; Martin, Juan J; Brandt, Tobias; Grond-Ginsbach, Caspar; Kloss, Manja; Mizutani, Tohru; Minematsu, Kazuo; Meschia, James F; Pereira, Vitor M; Bersano, Anna; Touzé, Emmanuel; Lyrer, Philippe A; Leys, Didier; Chabriat, Hugues; Markus, Hugh S; Worrall, Bradford B; Chabrier, Stéphane; Baumgartner, Ralph; Stapf, Christian; Tatlisumak, Turgut; Arnold, Marcel; Bousser, Marie-Germaine

    2015-06-01

    Spontaneous intracranial artery dissection is an uncommon and probably underdiagnosed cause of stroke that is defined by the occurrence of a haematoma in the wall of an intracranial artery. Patients can present with headache, ischaemic stroke, subarachnoid haemorrhage, or symptoms associated with mass effect, mostly on the brainstem. Although intracranial artery dissection is less common than cervical artery dissection in adults of European ethnic origin, intracranial artery dissection is reportedly more common in children and in Asian populations. Risk factors and mechanisms are poorly understood, and diagnosis is challenging because characteristic imaging features can be difficult to detect in view of the small size of intracranial arteries. Therefore, multimodal follow-up imaging is often needed to confirm the diagnosis. Treatment of intracranial artery dissections is empirical in the absence of data from randomised controlled trials. Most patients with subarachnoid haemorrhage undergo surgical or endovascular treatment to prevent rebleeding, whereas patients with intracranial artery dissection and cerebral ischaemia are treated with antithrombotics. Prognosis seems worse in patients with subarachnoid haemorrhage than in those without.

  13. Early and late management of type B aortic dissection.

    PubMed

    Nienaber, Christoph A; Divchev, Dimitar; Palisch, Holger; Clough, Rachel E; Richartz, Barbara

    2014-10-01

    The management of type B aortic dissection is undergoing profound changes with timely TEVAR accepted as first-line strategy in the setting of complicated dissection; with recent technological advances and in experienced hands this intervention is considered safe and life-saving. With the ability to remodel the dissected aorta as a result of scaffolding even pre-emptive endovascular treatment is being considered and supported by long-term stability and often prevention of aneurysmal expansion. This insight and a growing number of silent risk conditions (resistant hypertension, partial false lumen thrombosis) may lower the threshold for TEVAR in asymptomatic patients in the subacute phase. In the chronic phase of a type B dissection patients are usually free of symptoms, however, with the expanding false lumen at risk of rupture. Advanced TEVAR options (including branches and fenestrations) are likely to be used more often than open surgical replacement of such aneurysmatic segment of the dissected aorta in that chronic phase. All dissection patients should be offered lifelong surveillance.

  14. FBN1 Mutations in Patients With Descending Thoracic Aortic Dissections

    PubMed Central

    Brautbar, Ariel; LeMaire, Scott A.; Franco, Luis M.; Coselli, Joseph S.; Milewicz, Dianna M.; Belmont, John W.

    2013-01-01

    Aortic aneurysm and dissection cause significant morbidity and mortality. There are several known single gene disorders that predispose to isolated aortic disease and eventually aneurysm and dissection. FBN1 mutations are associated with multiple clinical phenotypes, including Marfan syndrome (MFS), MASS phenotype, and familial ectopia lentis, but rarely with isolated aortic aneurysm and dissection. In this report, we describe three patients who presented with primary descending thoracic aortic dissection and who were found to have an FBN1 mutation. None of the patients fulfilled clinical criteria for the diagnosis of MFS, and all had few or none of the skeletal features typical of the condition. Two patients had a history of long-term hypertension, and such a history was suspected in the third patient. These observations suggest that some individuals with FBN1 mutations have significant aortic disease involvement of other systems that is typical of FBN1 mutation-related syndromes. Superimposed risk factors, such as hypertension, may weaken the aortic wall and eventually lead to aortic dissection. Given that the cost continues to decrease, we suggest that diagnostic DNA sequencing for FBN1 mutations in patients with thoracic aortic aneurysms and dissection may be a practical clinical step in evaluating such patients and at-risk family members. PMID:20082464

  15. Three years of experience with a dissection table ventilation system.

    PubMed

    Martin, W D; Nemitz, J W; Hendley, A; Fisk, R M; Wells, J P

    1995-01-01

    A dissection table ventilation system that draws air across the cadaver and away from the table top was designed to fit the Shandon-Lipshaw AN-52 dissection table. Each U-shaped unit consists of a pair of hollow collection arms that attach to a collecting manifold at one end. During dissection the manifold is coupled to a central ventilation system through a flexible duct. The air from the table ventilation system is exhausted after passing through a heat recovery system. The unit is raised from the table surface during dissection of the body cavities to increase the efficiency of fume/odor removal. Eight hour exposure data for formaldehyde concentrations are presented. Data were collected from detectors positioned at selected levels above the cadaver during dissection, and above a tray on the table top containing a known volume of 4% formaldehyde or the West Virginia School of Osteopathic Medicine embalming fluid under varying airflow conditions. The results demonstrate that the table ventilation system is effective in reducing exposure to formaldehyde in the dissection laboratory.

  16. Rare Presentation of Left Lower Lobe Pulmonary Artery Dissection

    PubMed Central

    Hako, René; Fedačko, Ján; Morochovič, Radoslav; Kristian, Pavol; Pekárová, Tímea; Tuomainen, Petri; Pella, Daniel

    2017-01-01

    Background. Pulmonary arterial dissection with chronic pulmonary arterial hypertension as its major cause is a very rare but life-threatening condition. In most cases the main pulmonary trunk is the affected site usually without involvement of its branches. Segmental or lobar pulmonary artery dissection is extremely rare. Case Presentation. We report a unique case of left lower lobe pulmonary artery dissection in a 70-year-old male, with confirmed chronic pulmonary hypertension. To confirm dissection MDCT pulmonary angiography was used. Multiplanar reformation (MPR) images in sagittal, coronal, oblique sagittal, and curved projections were generated. This case report presents morphologic CT features of rare chronic left lobar pulmonary artery dissection associated with chronic pulmonary hypertension at a place of localised pulmonary artery calcification. CT pulmonary angiography excluded signs of thromboembolism and potential motion or flow artefacts. Conclusion. To the best of our knowledge, no case of lower lobe pulmonary artery dissection with flap calcification has been reported yet. CT imaging of the chest is a key diagnostic tool that is able to detect an intimal flap and a false lumen within the pulmonary arterial tree and is preferred in differential diagnosis of rare complications of sustained pulmonary arterial hypertension. PMID:28154579

  17. Endoscopic submucosal dissection for gastric adenomyoma

    PubMed Central

    Wang, Sinan; Cao, Hailong; Zhang, Yujie; Xu, Mengque; Chen, Xue; Piao, Meiyu; Wang, Bangmao

    2017-01-01

    Abstract Gastric adenomyoma (GA) is a kind of rare gastric submucosal eminence lesions. As the malignant transformation cannot be ruled out, surgery and laparoscopic resection are usually considered. The aim of this study is to evaluate the therapeutic effect and safety of endoscopic submucosal dissection (ESD) for GA. All of the patients with gastric submucosal eminence lesions who underwent ESD from June 2008 to June 2015 in General Hospital, Tianjin Medical University, China, were identified, and patients with GA, which was confirmed by pathological evaluation, were enrolled for further analysis. Among the 571 patients who received ESD, 15 cases with uncertain diagnosis before the procedure were finally confirmed as GA. The mean age of these 15 patients was 46.93 ± 15.56 years (range: 18–73). Most of the lesions were located in antrum (12/15 patients), with 2 in the body of stomach and 1 in cardia, respectively. The mean size of the lesions was 1.47 ± 0.67 cm (range: 0.4–3.0). According to the endoscopic ultrasonography, the lesions of 14 patients originated from submucosa and 1 originated from superficial muscularis, totally with mixed echoes changes. En bloc complete resection was achieved in all of the lesions. No perforation, intraoperative bleeding, delayed bleeding, and mortalities occurred. No recurrence or metastasis was found during 1 to 67 months. ESD appears to be a feasible, safe, and effective treatment for GA with clinical presentation of gastric submucosal eminence lesions. PMID:28248886

  18. Treatment of Ruptured Vertebral Artery Dissecting Aneurysms

    PubMed Central

    Hamasaki, Osamu; Ikawa, Fusao; Hidaka, Toshikazu; Kurokawa, Yasuharu; Yonezawa, Ushio

    2014-01-01

    Summary We evaluated the outcomes of endovascular or surgical treatment of ruptured vertebral artery dissecting aneurysms (VADAs), and investigated the relations between treatment complications and the development and location of the posterior inferior cerebellar artery (PICA). We treated 14 patients (12 men, two women; mean age, 56.2 years) with ruptured VADAs between March 1999 and June 2012 at our hospital. Six and eight patients had Hunt and Hess grades 1-3 and 4-5, respectively. Twelve patients underwent internal endovascular trapping, one underwent proximal endovascular occlusion alone, and one underwent proximal endovascular occlusion in the acute stage and occipital artery (OA)-PICA anastomosis and surgical trapping in the chronic stage. The types of VADA based on their location relative to the ipsilateral PICA were distal, PICA-involved, and non-PICA in nine, two, and three patients, respectively. The types of PICA based on their development and location were bilateral anterior inferior cerebellar artery (AICA)-PICA, ipsilateral AICA-PICA, extradural, and intradural type in one, two, two, and nine patients, respectively. Two patients with high anatomical risk developed medullary infarction, but their midterm outcomes were better than in previous reports. The modified Rankin scale indicated grades 0-2, 3-5, and 6 in eight, three, and three patients, respectively. A good outcome is often obtained in the treatment of ruptured VADA using internal endovascular trapping, except in the PICA-involved type, even with high-grade subarachnoid hemorrhage. Treatment of the PICA-involved type is controversial. The anatomical location and development of PICA may be predicted by complications with postoperative medullary infarction. PMID:24976093

  19. Circadian variation of acute aortic dissection.

    PubMed

    Seguchi, Masaru; Wada, Hiroshi; Sakakura, Kenichi; Nakagawa, Tom; Ibe, Tatsuro; Ikeda, Nahoko; Sugawara, Yoshitaka; Ako, Junya; Momomura, Shin-ichi

    2015-05-13

    Acute aortic dissection (AAD) is a life-threatening cardiovascular disease with high mortality. Hypertension is a well known risk factor of AAD. There have been previous reports about the association between circadian variation of blood pressure (BP) and cardiovascular events. However, little is known about the association between the onset-time of AAD and circadian variation of BP. The purpose of this study was to clarify the characteristics of circadian variation of BP in AAD and its relation to the onset-time of this disease. This study included type B spontaneous AAD patients who were referred to our institution and treated conservatively between January 2008 and June 2013. Patients with type A AAD, secondary to trauma, and type B AAD which preceded surgical intervention were excluded. Data were retrospectively collected from the hospital medical records. Sixty-eight patients with type B AAD were enrolled. The distribution of the circadian pattern in the study patients was as follows: extreme-dipper, 0% (none); dipper, 20.6% (n = 14); nondipper, 50% (n = 34); riser, 29.4% (n = 20). Non-dipper and riser patterns were more frequently observed compared with other population studies reported previously. Moreover, no patient in the dipper group had night-time onset while 31.5% of the patients in the absence of nocturnal BP fall group (non-dipper and riser) did (P = 0.01). Absence of a nocturnal BP fall was frequently seen in AAD patients. Absence of a nocturnal BP fall may be a risk factor of AAD. Circadian variation of BP may also affect the onset-time of type B AAD.

  20. Relative quantification of phosphoproteomic changes in grapevine (Vitis vinifera L.) leaves in response to abscisic acid

    PubMed Central

    Rattanakan, Supakan; George, Iniga; Haynes, Paul A; Cramer, Grant R

    2016-01-01

    In a previous transcriptomic analysis, abscisic acid (ABA) was found to affect the abundance of a number of transcripts in leaves of Cabernet Sauvignon grapevines with roots that had been exposed to 10 μm ABA for 2 h. Other work has indicated that ABA affects protein abundance and protein phosphorylation as well. In this study we investigated changes in protein abundance and phosphorylation of Cabernet Sauvignon grapevine leaves. Protein abundance was assessed by both label-free and isobaric-label quantitive proteomic methods. Each identified common proteins, but also additional proteins not found with the other method. Overall, several thousand proteins were identified and several hundred were quantified. In addition, hundreds of phosphoproteins were identified. Tens of proteins were found to be affected in the leaf after the roots had been exposed to ABA for 2 h, more than half of them were phosphorylated proteins. Many phosphosites were confirmed and several new ones were identified. ABA increased the abundance of some proteins, but the majority of the proteins had their protein abundance decreased. Many of these proteins were involved in growth and plant organ development, including proteins involved in protein synthesis, photosynthesis, sugar and amino-acid metabolism. This study provides new insights into how ABA regulates plant responses and acclimation to water deficits. PMID:27366326

  1. QUANTITATIVE MORPHOLOGY

    EPA Science Inventory

    Abstract: In toxicology, the role of quantitative assessment of brain morphology can be understood in the context of two types of treatment-related alterations. One type of alteration is specifically associated with treatment and is not observed in control animals. Measurement ...

  2. On the necessity of dissecting sequence similarity scores into segment-specific contributions for inferring protein homology, function prediction and annotation

    PubMed Central

    2014-01-01

    Background Protein sequence similarities to any types of non-globular segments (coiled coils, low complexity regions, transmembrane regions, long loops, etc. where either positional sequence conservation is the result of a very simple, physically induced pattern or rather integral sequence properties are critical) are pertinent sources for mistaken homologies. Regretfully, these considerations regularly escape attention in large-scale annotation studies since, often, there is no substitute to manual handling of these cases. Quantitative criteria are required to suppress events of function annotation transfer as a result of false homology assignments. Results The sequence homology concept is based on the similarity comparison between the structural elements, the basic building blocks for conferring the overall fold of a protein. We propose to dissect the total similarity score into fold-critical and other, remaining contributions and suggest that, for a valid homology statement, the fold-relevant score contribution should at least be significant on its own. As part of the article, we provide the DissectHMMER software program for dissecting HMMER2/3 scores into segment-specific contributions. We show that DissectHMMER reproduces HMMER2/3 scores with sufficient accuracy and that it is useful in automated decisions about homology for instructive sequence examples. To generalize the dissection concept for cases without 3D structural information, we find that a dissection based on alignment quality is an appropriate surrogate. The approach was applied to a large-scale study of SMART and PFAM domains in the space of seed sequences and in the space of UniProt/SwissProt. Conclusions Sequence similarity core dissection with regard to fold-critical and other contributions systematically suppresses false hits and, additionally, recovers previously obscured homology relationships such as the one between aquaporins and formate/nitrite transporters that, so far, was only

  3. Spontaneous vertebral dissection: Clinical, conventional angiographic, CT, and MR findings

    SciTech Connect

    Provenzale, J.M.; Morgenlander, J.C.; Gress, D.

    1996-03-01

    The purpose of this study was to determine if typical clinical and neuroradiologic patterns exist in patients with spontaneous vertebral artery (VA) dissection. The medical records and neuroradiologic examinations of 14 patients with spontaneous VA dissection were reviewed. The medical records were examined to exclude patients with a history of trauma and to record evidence of a nontratimatic precipitating event ({open_quotes}trivial trauma{close_quotes}) and presence of possible risk factors such as hypertension. All patients under-went conventional angiography, 13 either CT or MRI (II both CT and MRI), and 3 MRA. Conventional arteriograrris were evaluated for dissection site, evidence of fibromuscular dysplasia, luminal stenosis or occlusion, and pseudoaneurysm formation, CT examinations for the presence of infarction or subarachnoid hemorrhage, MR examinations for the presence of infarction or arterial signal abnormality, and MR angiograms for abnormality of the arterial signal column. Seven patients had precipitating events within 24 h of onset of symptoms that may have been causative of dissection and five had hypertension. At catheter angiography, two patients had dissections in two arteries (both VAs in one patient, VA and internal carotid artery in one patient), giving a total of 15 VAs with dissection. Dissection sites included V1 in four patients, V2 in one patient, V3 in three patients, V4 in six patients, and both V3 and V4 in one patient. Luminal stenosis was present in 13 VAs, occlusion in 2, pseudoaneurysm in 1, and evidence of fibromuscular dysplasia in 1. Posterior circulation infarcts were found on CT or MR in five patients. Subarachnoid hemorrhage was found on CT in two patients and by lumbar puncture alone in two patients. Abnormal periarterial signal on MRI was seen in three patients. MRA demonstrated absent VA signal in one patient, pseudoaneurysm in one, and a false-negative examination in one.

  4. Anatomy-Specific Virtual Reality Simulation in Temporal Bone Dissection.

    PubMed

    Locketz, Garrett D; Lui, Justin T; Chan, Sonny; Salisbury, Kenneth; Dort, Joseph C; Youngblood, Patricia; Blevins, Nikolas H

    2017-03-01

    Objective To evaluate the effect of anatomy-specific virtual reality (VR) surgical rehearsal on surgeon confidence and temporal bone dissection performance. Study Design Prospective pre- and poststudy of a novel virtual surgical rehearsal platform. Setting Academic otolaryngology-head and neck surgery residency training programs. Subjects and Methods Sixteen otolaryngology-head and neck surgery residents from 2 North American training institutions were recruited. Surveys were administered to assess subjects' baseline confidence in performing 12 subtasks of cortical mastoidectomy with facial recess. A cadaver temporal bone was randomly assigned to each subject. Cadaver specimens were scanned with a clinical computed tomography protocol, allowing the creation of anatomy-specific models for use in a VR surgical rehearsal platform. Subjects then rehearsed a virtual mastoidectomy on data sets derived from their specimens. Surgical confidence surveys were administered again. Subjects then dissected assigned cadaver specimens, which were blindly graded with a modified Welling scale. A final survey assessed the perceived utility of rehearsal on dissection performance. Results Of 16 subjects, 14 (87.5%) reported a significant increase in overall confidence after conducting an anatomy-specific VR rehearsal. A significant correlation existed between perceived utility of rehearsal and confidence improvement. The effect of rehearsal on confidence was dependent on trainee experience and the inherent difficulty of the surgical subtask. Postrehearsal confidence correlated strongly with graded dissection performance. Subjects rated anatomy-specific rehearsal as having a moderate to high contribution to their dissection performance. Conclusion Anatomy-specific virtual rehearsal improves surgeon confidence in performing mastoid dissection, dependent on surgeon experience and task difficulty. The subjective confidence gained through rehearsal correlates positively with subsequent

  5. A comparison of retention of anatomical knowledge in an introductory college biology course: Traditional dissection vs. virtual dissection

    NASA Astrophysics Data System (ADS)

    Taeger, Kelli Rae

    Dissection has always played a crucial role in biology and anatomy courses at all levels of education. However, in recent years, ethical concerns, as well as improved technology, have brought to the forefront the issue of whether virtual dissection is as effective or whether it is more effective than traditional dissection. Most prior research indicated the two methods produced equal results. However, none of those studies examined retention of information past the initial test of knowledge. Two groups of college students currently enrolled in an introductory level college biology course were given one hour to complete a frog dissection. One group performed a traditional frog dissection, making cuts in an actual preserved frog specimen with scalpels and scissors. The other group performed a virtual frog dissection, using "The Digital Frog 2" software. Immediately after the dissections were completed, each group was given an examination consisting of questions on actual specimens, pictures generated from the computer software, and illustrations that neither group had seen. Two weeks later, unannounced, the groups took the same exam in order to test retention. The traditional dissection group scored significantly higher on two of the three sections, as well as the total score on the initial exam. However, with the exception of specimen questions (on which the traditional group retained significantly more information), there was no significant difference in the retention from exam 1 to exam 2 between the two groups. These results, along with the majority of prior studies, show that the two methods produce, for the most part, the same end results. Therefore, the decision of which method to employ should be based on the goals and preferences of the instructor(s) and the department. If that department's goals include: Being at the forefront of new technology, increasing time management, increasing student: teacher ratio for economic reasons, and/or ethical issues, then

  6. Quantitative glycomics.

    PubMed

    Orlando, Ron

    2010-01-01

    The ability to quantitatively determine changes is an essential component of comparative glycomics. Multiple strategies are available by which this can be accomplished. These include label-free approaches and strategies where an isotopic label is incorporated into the glycans prior to analysis. The focus of this chapter is to describe each of these approaches while providing insight into their strengths and weaknesses, so that glycomic investigators can make an educated choice of the strategy that is best suited for their particular application.

  7. Comparative N-Glycoproteomic and Phosphoproteomic Profiling of Human Placental Plasma Membrane between Normal and Preeclampsia Pregnancies with High-Resolution Mass Spectrometry

    PubMed Central

    Wang, Fuqiang; Wang, Ling; Shi, Zhonghua; Liang, Gaolin

    2013-01-01

    Preeclampsia is a serious complication of pregnancy, which affects 2–8% of all pregnancies and is one of the leading causes of maternal and perinatal mortality and morbidity worldwide. To better understand the molecular mechanisms involved in pathological development of placenta in preeclampsia, we used high-resolution LC-MS/MS technologies to construct a comparative N-glycoproteomic and phosphoproteomic profiling of human placental plasma membrane in normal and preeclamptic pregnancies. A total of 1027 N-glyco- and 2094 phospho- sites were detected in human placental plasma membrane, and 5 N-glyco- and 38 phospho- proteins, respectively, with differentially expression were definitively identified between control and preeclamptic placental plasma membrane. Further bioinformatics analysis indicated that these differentially expressed proteins correlate with several specific cellular processes occurring during pathological changes of preeclamptic placental plasma membrane. PMID:24260401

  8. Comparative Phosphoproteomics Reveals the Role of AmpC β-lactamase Phosphorylation in the Clinical Imipenem-resistant Strain Acinetobacter baumannii SK17*

    PubMed Central

    Lai, Juo-Hsin; Yang, Jhih-Tian; Chern, Jeffy; Chen, Te-Li; Wu, Wan-Ling; Liao, Jiahn-Haur; Tsai, Shih-Feng; Liang, Suh-Yuen; Chou, Chi-Chi

    2016-01-01

    Nosocomial infectious outbreaks caused by multidrug-resistant Acinetobacter baumannii have emerged as a serious threat to human health. Phosphoproteomics of pathogenic bacteria has been used to identify the mechanisms of bacterial virulence and antimicrobial resistance. In this study, we used a shotgun strategy combined with high-accuracy mass spectrometry to analyze the phosphoproteomics of the imipenem-susceptible strain SK17-S and -resistant strain SK17-R. We identified 410 phosphosites on 248 unique phosphoproteins in SK17-S and 285 phosphosites on 211 unique phosphoproteins in SK17-R. The distributions of the Ser/Thr/Tyr/Asp/His phosphosites in SK17-S and SK17-R were 47.0%/27.6%/12.4%/8.0%/4.9% versus 41.4%/29.5%/17.5%/6.7%/4.9%, respectively. The Ser-90 phosphosite, located on the catalytic motif S88VS90K of the AmpC β-lactamase, was first identified in SK17-S. Based on site-directed mutagenesis, the nonphosphorylatable mutant S90A was found to be more resistant to imipenem, whereas the phosphorylation-simulated mutant S90D was sensitive to imipenem. Additionally, the S90A mutant protein exhibited higher β-lactamase activity and conferred greater bacterial protection against imipenem in SK17-S compared with the wild-type. In sum, our results revealed that in A. baumannii, Ser-90 phosphorylation of AmpC negatively regulates both β-lactamase activity and the ability to counteract the antibiotic effects of imipenem. These findings highlight the impact of phosphorylation-mediated regulation in antibiotic-resistant bacteria on future drug design and new therapies. PMID:26499836

  9. Differences in Beef Quality between Angus (Bos taurus taurus) and Nellore (Bos taurus indicus) Cattle through a Proteomic and Phosphoproteomic Approach

    PubMed Central

    Chizzotti, Mario Luiz; Vital, Camilo Elber; Baracat-Pereira, Maria Cristina; Barros, Edvaldo; Busato, Karina Costa; Gomes, Rafael Aparecido; Ladeira, Márcio Machado; Martins, Taiane da Silva

    2017-01-01

    Proteins are the major constituents of muscle and are key molecules regulating the metabolic changes during conversion of muscle to meat. Brazil is one of the largest exporters of beef and most Brazilian cattle are composed by zebu (Nellore) genotype. Bos indicus beef is generally leaner and tougher than Bos taurus such as Angus. The aim of this study was to compare the muscle proteomic and phosphoproteomic profile of Angus and Nellore. Seven animals of each breed previously subjected the same growth management were confined for 84 days. Proteins were extracted from Longissimus lumborum samples collected immediately after slaughter and separated by two-dimensional electrophoresis. Pro-Q Diamond stain was used in phosphoproteomics. Proteins identification was performed using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Tropomyosin alpha-1 chain, troponin-T, myosin light chain-1 fragment, cytoplasmic malate dehydrogenase, alpha-enolase and 78 kDa glucose-regulated protein were more abundant in Nellore, while myosin light chain 3, prohibitin, mitochondrial stress-70 protein and heat shock 70 kDa protein 6 were more abundant in Angus (P<0.05). Nellore had higher phosphorylation of myosin regulatory light chain-2, alpha actin-1, triosephosphate isomerase and 14-3-3 protein epsilon. However, Angus had greater phosphorylation of phosphoglucomutase-1 and troponin-T (P<0.05). Therefore, proteins involved in contraction and muscle organization, myofilaments expressed in fast or slow-twitch fibers and heat shock proteins localized in mitochondria or sarcoplasmic reticulum and involved in cell flux of calcium and apoptosis might be associated with differences in beef quality between Angus and Nellore. Furthermore, prohibitin appears to be a potential biomarker of intramuscular fat in cattle. Additionally, differences in phosphorylation of myofilaments and glycolytic enzymes could be involved with differences in muscle contraction force

  10. Unraveling pancreatic islet biology by quantitative proteomics

    SciTech Connect

    Zhou, Jianying; Dann, Geoffrey P.; Liew, Chong W.; Smith, Richard D.; Kulkarni, Rohit N.; Qian, Weijun

    2011-08-01

    The pancreatic islets of Langerhans play a critical role in maintaining blood glucose homeostasis by secreting insulin and several other important peptide hormones. Impaired insulin secretion due to islet dysfunction is linked to the pathogenesis underlying both Type 1 and Type 2 diabetes. Over the past 5 years, emerging proteomic technologies have been applied to dissect the signaling pathways that regulate islet functions and gain an understanding of the mechanisms of islet dysfunction relevant to diabetes. Herein, we briefly review some of the recent quantitative proteomic studies involving pancreatic islets geared towards gaining a better understanding of islet biology relevant to metabolic diseases.

  11. Genetic dissection of maize phenology using an intraspecific introgression library

    PubMed Central

    2011-01-01

    Background Collections of nearly isogenic lines where each line carries a delimited portion of a donor source genome into a common recipient genetic background are known as introgression libraries and have already shown to be instrumental for the dissection of quantitative traits. By means of marker-assisted backcrossing, we have produced an introgression library using the extremely early-flowering maize (Zea mays L.) variety Gaspé Flint and the elite line B73 as donor and recipient genotypes, respectively, and utilized this collection to investigate the genetic basis of flowering time and related traits of adaptive and agronomic importance in maize. Results The collection includes 75 lines with an average Gaspé Flint introgression length of 43.1 cM. The collection was evaluated for flowering time, internode length, number of ears, number of nodes (phytomeres), number of nodes above the ear, number and proportion of nodes below the ear and plant height. Five QTLs for flowering time were mapped, all corresponding to major QTLs for number of nodes. Three additional QTLs for number of nodes were mapped. Besides flowering time, the QTLs for number of nodes drove phenotypic variation for plant height and number of nodes below and above the top ear, but not for internode length. A number of apparently Mendelian-inherited phenotypes were also observed. Conclusions While the inheritance of flowering time was dominated by the well-known QTL Vgt1, a number of other important flowering time QTLs were identified and, thanks to the type of plant material here utilized, immediately isogenized and made available for fine mapping. At each flowering time QTL, early flowering correlated with fewer vegetative phytomeres, indicating the latter as a key developmental strategy to adapt the maize crop from the original tropical environment to the northern border of the temperate zone (southern Canada), where Gaspé Flint was originally cultivated. Because of the trait differences

  12. Dissection of a nuclear localization signal.

    PubMed

    Hodel, M R; Corbett, A H; Hodel, A E

    2001-01-12

    The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein import receptors. This study seeks to decipher the energetic details of NLS recognition by the receptor importin alpha through quantitative analysis of variant NLSs. The relative importance of each residue in two monopartite NLS sequences was determined using an alanine scanning approach. These measurements yield an energetic definition of a monopartite NLS sequence where a required lysine residue is followed by two other basic residues in the sequence K(K/R)X(K/R). In addition, the energetic contributions of the second basic cluster in a bipartite NLS ( approximately 3 kcal/mol) as well as the energy of inhibition of the importin alpha importin beta-binding domain ( approximately 3 kcal/mol) were also measured. These data allow the generation of an energetic scale of nuclear localization sequences based on a peptide's affinity for the importin alpha-importin beta complex. On this scale, a functional NLS has a binding constant of approximately 10 nm, whereas a nonfunctional NLS has a 100-fold weaker affinity of 1 microm. Further correlation between the current in vitro data and in vivo function will provide the foundation for a comprehensive quantitative model of protein import.

  13. Advanced, recurrent mesothelioma growth mimicking an aortic dissection.

    PubMed

    Pankhania, Miran; Hardiment, Kate; Marathe, Mandar

    2011-02-02

    In the emergency setting, a cold, clammy, dyspnoeic patient presenting with interscapular chest pain and unequal blood pressures suggests an acute aortic dissection until proven otherwise. By means of a case report, the authors detail one such patient who presented identically to one having an acute aortic dissection. Initial assessment showed unequal blood pressures in left and right arms, a resting tachycardia and indistinct heart sounds. Fluid resuscitation failed to improve the patient's physiological parameters and they rapidly deteriorated. The medical history included mesothelioma and atrial fibrillation. Existing investigations were reviewed and after thorough consideration of the patient's premorbid state and likely prognosis, the decision was made to palliate. The patient died shortly after being transferred to the oncology ward. Imaging is therefore integral to the assessment and management of a patient in whom an aortic dissection is feared.

  14. Proteomic approaches to dissect platelet function: half the story

    PubMed Central

    Gnatenko, Dmitri V.; Perrotta, Peter L.; Bahou, Wadie F.

    2006-01-01

    Platelets play critical roles in diverse hemostatic and pathologic disorders and are broadly implicated in various biological processes that include inflammation, wound healing, and thrombosis. Recent progress in high-throughput mRNA and protein profiling techniques has advanced our understanding of the biological functions of platelets. Platelet proteomics has been adopted to decode the complex processes that underlie platelet function by identifying novel platelet-expressed proteins, dissecting mechanisms of signal or metabolic pathways, and analyzing functional changes of the platelet proteome in normal and pathologic states. The integration of transcriptomics and proteomics, coupled with progress in bioinformatics, provides novel tools for dissecting platelet biology. In this review, we focus on current advances in platelet proteomic studies, with emphasis on the importance of parallel transcriptomic studies to optimally dissect platelet function. Applications of these global profiling approaches to investigate platelet genetic diseases and platelet-related disorders are also addressed. PMID:16926286

  15. Aortic dissection accompanied by preeclampsia in a postpartum young woman

    PubMed Central

    Park, Jin-Wan; Kim, Su-Mi; Yu, Gyu-Bong

    2016-01-01

    Aortic dissection is very rare in obstetrics, but it is a fatal disease. A 37-weeks primigravida woman with dyspnea and pitting edema presented to our emergency room. The patient was diagnosed with preeclampsia and underwent an emergency cesarean section under spinal anesthesia. The patient complained of severe dyspnea after the cesarean section, and the chest computed tomography scan was done. With the finding of aortic dissection, cardiopulmonary arrest occurred 5 hours after the cesarean section, and the patient died without reaction to cardio-pulmonary resuscitation. If a patient with preeclampsia complains of severe dyspnea or chest pain, aortic dissection needs to be suspected and a diagnosis should not be delayed. PMID:27668205

  16. Lymphoplasmacytic aortitis and acute aortic dissection. An uncommon association.

    PubMed

    Faye-Petersen, O M; Arnold, M M; Grizzle, W E; Lie, J T

    1996-04-01

    A 43-year-old white man with a history of cigarette smoking, hypertension, nephrolithiasis, and cervical degenerative arthritis was hospitalized for sudden-onset severe, substernal, and pleuritic chest pain with epigastric radiation. Despite evaluation, the cause remained unclear and the patient expired on hospital day 5. Autopsy revealed acute Stanford type A aortic dissection, hemopericardium, and hemothorax. Grossly, the aorta and its branches, including uninvolved medium-sized arteries, displayed extreme mural fragility. Microscopic examination showed a primary lymphoplasmacytic aortitis-periaortitis without giant cells. Rents within the tunica media, medial-adventitial inflammation, and elastic fiber disruption were limited to sites of gross aortic dissection. Muscular arteries showed patchy, chronic arteritis-periarteritis without giant cell infiltrate or aneurysm formation. This case documents an unusual association of primary lymphoplasmacytic aortitis and aortic dissection.

  17. Free manual of cadaver dissection modifiable by other anatomists.

    PubMed

    Chung, Beom Sun; Chung, Min Suk

    2015-06-01

    Even in the rapidly changing field of cadaver dissection, published guide books still play an important role in the anatomy lab. However, commercial manuals with lengthy volumes and inflexible copyrights have several limitations which can be complemented by open-source manuals. Recently, the authors have manufactured and distributed a free electronic dissection manual (anatomy.co.kr), where descriptions are written concisely and images are drawn schematically. Moreover, simplified signs are employed to represent the cadaver viewing angles and manner of dissection. Based on the original files of this manual, other anatomists can revise and utilize the descriptions and figures. We expect many updated versions of our manual to be shared between students all over the world.

  18. Digital dissection system for medical school anatomy training

    NASA Astrophysics Data System (ADS)

    Augustine, Kurt E.; Pawlina, Wojciech; Carmichael, Stephen W.; Korinek, Mark J.; Schroeder, Kathryn K.; Segovis, Colin M.; Robb, Richard A.

    2003-05-01

    As technology advances, new and innovative ways of viewing and visualizing the human body are developed. Medicine has benefited greatly from imaging modalities that provide ways for us to visualize anatomy that cannot be seen without invasive procedures. As long as medical procedures include invasive operations, students of anatomy will benefit from the cadaveric dissection experience. Teaching proper technique for dissection of human cadavers is a challenging task for anatomy educators. Traditional methods, which have not changed significantly for centuries, include the use of textbooks and pictures to show students what a particular dissection specimen should look like. The ability to properly carry out such highly visual and interactive procedures is significantly constrained by these methods. The student receives a single view and has no idea how the procedure was carried out. The Department of Anatomy at Mayo Medical School recently built a new, state-of-the-art teaching laboratory, including data ports and power sources above each dissection table. This feature allows students to access the Mayo intranet from a computer mounted on each table. The vision of the Department of Anatomy is to replace all paper-based resources in the laboratory (dissection manuals, anatomic atlases, etc.) with a more dynamic medium that will direct students in dissection and in learning human anatomy. Part of that vision includes the use of interactive 3-D visualization technology. The Biomedical Imaging Resource (BIR) at Mayo Clinic has developed, in collaboration with the Department of Anatomy, a system for the control and capture of high resolution digital photographic sequences which can be used to create 3-D interactive visualizations of specimen dissections. The primary components of the system include a Kodak DC290 digital camera, a motorized controller rig from Kaidan, a PC, and custom software to synchronize and control the components. For each dissection procedure, the

  19. Dissection of the atrial wall after mitral valve replacement.

    PubMed Central

    Lukács, L; Kassai, I; Lengyel, M

    1996-01-01

    We describe an unusual sequela of mitral valve replacement in a 50-year-old woman who had undergone a closed mitral commissurotomy in 1975. She was admitted to our hospital because of mitral restenosis in November 1993, at which time her mitral valve was replaced with a mechanical prosthesis. On the 8th postoperative day, the patient developed symptoms of heart failure; transesophageal echocardiography revealed dissection and rupture of the left atrial wall. At prompt reoperation, we found an interlayer dissection and rupture of the atrial wall into the left atrium. We repaired the ruptured atrial wall with a prosthetic patch. The postoperative course was uneventful, and postoperative transesophageal echocardiography showed normal prosthetic valve function and no dissection. Images PMID:8680278

  20. Are all hands-on activities equally effective? Effect of using plastic models, organ dissections, and virtual dissections on student learning and perceptions.

    PubMed

    Lombardi, Sara A; Hicks, Reimi E; Thompson, Katerina V; Marbach-Ad, Gili

    2014-03-01

    This study investigated the impact of three commonly used cardiovascular model-assisted activities on student learning and student attitudes and perspectives about science. College students enrolled in a Human Anatomy and Physiology course were randomly assigned to one of three experimental groups (organ dissections, virtual dissections, or plastic models). Each group received a 15-min lecture followed by a 45-min activity with one of the treatments. Immediately after the lesson and then 2 mo later, students were tested on anatomy and physiology knowledge and completed an attitude survey. Students who used plastic models achieved significantly higher overall scores on both the initial and followup exams than students who performed organ or virtual dissections. On the initial exam, students in the plastic model and organ dissection treatments scored higher on anatomy questions than students who performed virtual dissections. Students in the plastic model group scored higher than students who performed organ dissections on physiology questions. On the followup exam, when asked anatomy questions, students in the plastic model group scored higher than dissection students and virtual dissection students. On attitude surveys, organ dissections had higher perceived value and were requested for inclusion in curricula twice as often as any other activity. Students who performed organ dissections were more likely than the other treatment groups to agree with the statement that "science is fun," suggesting that organ dissections may promote positive attitudes toward science. The findings of this study provide evidence for the importance of multiple types of hands-on activities in anatomy laboratory courses.

  1. Case Report and Review of Literature: Late Retrograde Type A Aortic Dissection With Rupture after Repair of Type B Aortic Dissection with a GORE TAG Endovascular Prosthesis.

    PubMed

    Manetta, Frank; Ajakaiye, Bayo; Scheinerman, S Jacob; Yu, Pey-Jen

    2014-06-01

    Acute aortic dissection is the most common catastrophic condition of the aorta. Treatment options include open surgery and thoracic endovascular aortic reconstruction (TEVAR). We present a late Type A dissection as a complication of the management of descending aortic dissections with TEVAR and a review of the literature. TEVAR of the thoracic aorta is a viable treatment option for the management of complicated descending thoracic aortic dissections. Careful patient selection is necessary as medical therapy successfully treats the majority of uncomplicated Type B dissections. TEVAR should be reserved for patients with complicated Type B dissections or those who fail nonoperative management. Close postoperative monitoring is necessary when TEVAR is performed and should be accompanied by lifelong surveillance. A high level of suspicion is important to identify retrograde Type A dissections in these patients given its rarity and the ambiguity of its clinical presentation.

  2. Dissection of brassinosteroid-regulated proteins in rice embryos during germination by quantitative proteomics

    PubMed Central

    Li, Qian-Feng; Xiong, Min; Xu, Peng; Huang, Li-Chun; Zhang, Chang-Quan; Liu, Qiao-Quan

    2016-01-01

    Brassinosteroids (BRs), essential plant-specific steroidal hormones, function in a wide spectrum of plant growth and development events, including seed germination. Rice is not only a monocotyledonous model plant but also one of the most important staple food crops of human beings. Rice seed germination is a decisive event for the next-generation of plant growth and successful seed germination is critical for rice yield. However, little is known about the molecular mechanisms on how BR modulates seed germination in rice. In the present study, we used isobaric tags for relative and absolute quantification (iTRAQ) based proteomic approach to study BR-regulated proteome during the early stage of seed germination. The results showed that more than 800 BR-responsive proteins were identified, including 88 reliable target proteins responsive to stimuli of both BR-deficiency and BR-insensitivity. Moreover, 90% of the 88 target proteins shared a similar expression change pattern. Gene ontology and string analysis indicated that ribosomal structural proteins, as well as proteins involved in protein biosynthesis and carbohydrate metabolisms were highly clustered. These findings not only enrich BR-regulated protein database in rice seeds, but also allow us to gain novel insights into the molecular mechanism of BR regulated seed germination. PMID:27703189

  3. Dissecting sources of quantitative gene expression pattern divergence between Drosophila species

    PubMed Central

    Wunderlich, Zeba; Bragdon, Meghan D; Eckenrode, Kelly B; Lydiard-Martin, Tara; Pearl-Waserman, Sivanne; DePace, Angela H

    2012-01-01

    Gene expression patterns can diverge between species due to changes in a gene's regulatory DNA or changes in the proteins, e.g., transcription factors (TFs), that regulate the gene. We developed a modeling framework to uncover the sources of expression differences in blastoderm embryos of three Drosophila species, focusing on the regulatory circuit controlling expression of the hunchback (hb) posterior stripe. Using this framework and cellular-resolution expression measurements of hb and its regulating TFs, we found that changes in the expression patterns of hb's TFs account for much of the expression divergence. We confirmed our predictions using transgenic D. melanogaster lines, which demonstrate that this set of orthologous cis-regulatory elements (CREs) direct similar, but not identical, expression patterns. We related expression pattern differences to sequence changes in the CRE using a calculation of the CRE's TF binding site content. By applying this calculation in both the transgenic and endogenous contexts, we found that changes in binding site content affect sensitivity to regulating TFs and that compensatory evolution may occur in circuit components other than the CRE. PMID:22893002

  4. Dissecting sources of quantitative gene expression pattern divergence between Drosophila species.

    PubMed

    Wunderlich, Zeba; Bragdon, Meghan D; Eckenrode, Kelly B; Lydiard-Martin, Tara; Pearl-Waserman, Sivanne; DePace, Angela H

    2012-01-01

    Gene expression patterns can diverge between species due to changes in a gene's regulatory DNA or changes in the proteins, e.g., transcription factors (TFs), that regulate the gene. We developed a modeling framework to uncover the sources of expression differences in blastoderm embryos of three Drosophila species, focusing on the regulatory circuit controlling expression of the hunchback (hb) posterior stripe. Using this framework and cellular-resolution expression measurements of hb and its regulating TFs, we found that changes in the expression patterns of hb's TFs account for much of the expression divergence. We confirmed our predictions using transgenic D. melanogaster lines, which demonstrate that this set of orthologous cis-regulatory elements (CREs) direct similar, but not identical, expression patterns. We related expression pattern differences to sequence changes in the CRE using a calculation of the CRE's TF binding site content. By applying this calculation in both the transgenic and endogenous contexts, we found that changes in binding site content affect sensitivity to regulating TFs and that compensatory evolution may occur in circuit components other than the CRE.

  5. Superior Mesenteric Artery Dissection after Extracorporeal Shockwave Lithotripsy

    PubMed Central

    Bakoyiannis, Christos; Anastasiou, Ioannis; Koutsoumpelis, Andreas; Fragiadis, Evangelos; Felesaki, Eleni; Kafeza, Marina; Georgopoulos, Sotirios; Tsigris, Christos

    2012-01-01

    The use of shockwave lithotripsy is currently the mainstay of treatment in renal calculosis. Several complications including vessel injuries have been implied to extracorporeal shockwave lithotripsy. We report an isolated dissection of the superior mesenteric artery in a 60-year-old male presenting with abdominal pain which occurred three days after extracorporeal shockwave lithotripsy. The patient was treated conservatively and the abdominal pain subsided 24 hours later. The patient's history, the course of his disease, and the timing may suggest a correlation between the dissection and the ESWL. PMID:23304627

  6. Vertebral artery dissection related to basilar impression: case report.

    PubMed

    Dickinson, L D; Tuite, G F; Colon, G P; Papadopoulos, S M

    1995-04-01

    A 50-year-old man with myelopathy secondary to basilar impression developed bilateral vertebral artery dissection after undergoing treatment with 8 pounds of cervical traction. The vertebral artery dissection resulted in vertebrobasilar insufficiency and posterior circulation stroke. In this report, the current management philosophies in the treatment of basilar impression are discussed, and the pertinent neurovascular anatomy is illustrated. This report suggests that vertebral artery injury may result from attempted reduction of severe basilar impression. Regardless of the cause of cranial settling, the risk of vertebral artery injury with cervical traction should be considered in patients with severe basilar impression.

  7. Spontaneous dissections of multiple visceral arteries: an extremely rare case

    PubMed Central

    Le, Trong Binh; Hong, Kee Chun; Cho, Soon Gu; Park, Keun-Myoung

    2017-01-01

    Spontaneous dissections of visceral arteries without aortic involvement are very rare. The etiologies of these entities are unclear and their clinical managements remain controversial. We report a case of spontaneous multiple dissections affecting 4 visceral arteries including the superior mesenteric artery, the celiac artery and the bilateral renal arteries. The patient was managed conservatively and endovascularly. The clinical manifestation markedly improved and laboratory tests returned to normal limits within 1 week. The regular follow-up suggested a good clinical and radiological outcome until 84 months. PMID:28382296

  8. Acute Aortic Dissection in Third Trimester Pregnancy without Risk Factors

    PubMed Central

    Kinney-Ham, Lisa; Nguyen, H. Bryant; Steele, Robert; Walters, Elizabeth L

    2011-01-01

    Spontaneous aortic dissection in pregnancy is rare and life threatening for both the mother and the fetus. Most commonly, it is associated with connective tissue disorders, cardiac valve variants, or trauma. We present the case of a 23-year-old previously healthy woman, 36 weeks pregnant with a syncopal episode after dyspnea and vomiting. She subsequently developed cardiac arrest and underwent aggressive resuscitation, emergent thoracotomy, and cesarean delivery without recovery. On autopsy, she was found to have an aortic dissection of the ascending aorta. This case is presented to raise awareness and review the literature and the clinical approach to critical care for pregnant patients. PMID:22224164

  9. Determining cell division symmetry through the dissection of dividing cells using single-cell expression analysis.

    PubMed

    Jasnos, Lukasz; Sawado, Tomoyuki

    2014-03-01

    Symmetric cell divisions give rise to two sister cells that are identical to each other, whereas asymmetric divisions produce two sister cells with distinctive phenotypes. Although cell division symmetry is usually determined on the basis of a few markers or biological functions, the overall similarity between sister cells has not been thoroughly examined at a molecular level. Here we provide a protocol to separate sister embryonic stem cells (ESCs) and to conduct multiplexed gene expression analyses at the single-cell level by using 48 ESC genes. The procedure includes the dissection of dividing, paired sister cells by micromanipulation, followed by cell lysis, reverse transcription, gene-specific cDNA amplification and multiplexed quantitative PCR analyses. This protocol can be completed in 10 d, and it can be readily adapted to other cell types that are able to grow in suspension culture.

  10. Role of cadaveric dissections in modern medical curricula: a study on student perceptions

    PubMed Central

    Nirthanan, Selvanayagam N.; Khoo, Tien K.; Tedman, Raymond

    2015-01-01

    The shift from traditional medical curricula to newer teaching and learning approaches such as problem-based learning has often resulted in omission or significant reduction of cadaveric dissections as a method of learning anatomy. The objective of this study was to evaluate students' perception of dissection in a graduate-entry, problem-based learning-based medical curriculum. At the end of the musculoskeletal dissection program in second year, a Likert-type questionnaire was used to explore medical student perceptions of the perceived advantages and challenges of cadaveric dissections in comparison with other anatomy teaching methods. Overall, a majority of students had a positive perception of dissections. Students who attended dissections regularly had significantly more positive perceptions about their experience and were in agreement with statements such as "dissections make learning more interesting" and "I would be disadvantaged if I did not attend dissection classes." Non-regular attendance was associated with statements about dissections such as "I do not like the smell," "time consuming," and "bored with the way it is carried-out." A follow-up study after completion of the medical program revealed a significant improvement of positive perception about dissection. Student perceptions appear to favour a role for cadaveric dissection in learning anatomy in modern medical curricula. However, optimal and effective integration of dissections is important, with consideration given to its structure and extent of content weighed against logistics and availability of resources; while addressing negative perceptions of dissection-based teaching. PMID:26417481

  11. Exploring the human leukocyte phosphoproteome using a microfluidic reversed-phase-TiO2-reversed-phase high-performance liquid chromatography phosphochip coupled to a quadrupole time-of-flight mass spectrometer.

    PubMed

    Raijmakers, Reinout; Kraiczek, Karsten; de Jong, Ad P; Mohammed, Shabaz; Heck, Albert J R

    2010-02-01

    The study of protein phosphorylation events is one of the most important challenges in proteome analysis. Despite the importance of phosphorylation for many regulatory processes in cells and many years of phosphoprotein and phosphopeptide research, the identification and characterization of phosphorylation by mass spectrometry is still a challenging task. Recently, we introduced an approach that facilitates the analysis of phosphopeptides by performing automated, online, TiO(2) enrichment of phosphopeptides prior to mass spectrometry (MS) analysis. The implementation of that method on a "plug-and-play" microfluidic high-performance liquid chromatography (HPLC) chip design will potentially open up efficient phosphopeptide enrichment methods enabling phosphoproteomics analyses by a broader research community. Following our initial proof of principle, whereby the device was coupled to an ion trap, we now show that this so-called phosphochip is capable of the enrichment of large numbers of phosphopeptides from complex cellular lysates, which can be more readily identified when coupled to a higher resolution quadrupole time-of-flight (Q-TOF) mass spectrometer. We use the phosphochip-Q-TOF setup to explore the phosphoproteome of nonstimulated primary human leukocytes where we identify 1012 unique phosphopeptides corresponding to 960 different phosphorylation sites providing for the first time an overview of the phosphoproteome of these important circulating white blood cells.

  12. Combining classical trait and microarray data to dissect transcriptional regulation: a case study.

    PubMed

    Wang, Dong; Nettleton, Dan

    2008-03-01

    The selective transcriptional profiling approach involves selecting an optimal subset of individuals to microarray from a larger set of individuals for which relatively inexpensive quantitative trait and molecular marker data are available. The goal of the selection and subsequent analyses is to identify genes whose expression is associated with a quantitative trait or quantitative trait locus (QTL). In this paper, we applied the selective transcriptional profiling approach to data sets concerning flowering time and gene transcription levels of Arabidopsis recombinant inbred lines. Our results confirm that the selective transcriptional profiling approach can achieve much greater power for uncovering associations than standard approaches that ignore information from classical traits. In addition, we show that selective transcriptional profiling can achieve power similar to standard approaches at a fraction of the cost and effort. We also identified three groups of genes which show distinctive patterns with regard to gene expression levels, QTL genotype, and a classical trait. This study represents the first application of selective transcriptional profiling to real data and serves as a template for dissecting gene regulation networks related to a classical trait using the selective transcriptional profiling approach.

  13. [Spontaneous renal artery dissection with renal infarction: a case report].

    PubMed

    Oki, Takashi; Adachi, Hiroyuki; Tahara, Hideo; Kino, Sigeo

    2011-11-01

    A 58-year-old woman visited our hospital with nausea and right flank pain. At first abdominal ultrasonography was performed, suggesting a right renal infarction. Computed tomography (CT) study of the abdomen with intravenous contrast was performed to determine the cause of the symptoms. The scan revealed poor enhancement in the lower half of the right kidney. She was diagnosed with a right renal infarction. She was initially treated with anticoagulant therapy, but 5 days later, she complained of nausea. This time, CT demonstrated exacerbation of a right renal infarction with renal artery dissection. Based on this finding, we performed a right nephrectomy. The result of pathology was segmental arterial mediolysis. She was discharged 12 days after the surgery and is doing well at 6 months after discharge. Spontaneous renal artery dissection is a rare disease. It constitutes approximately 0.05% of arteriographic dissections. In addition, spontaneous renal artery dissection shows nonspecific symptoms. Together, these two factors may cause a delay in diagnosis.

  14. Dissecting Situational Strength: Theoretical Analysis and Empirical Tests

    DTIC Science & Technology

    2012-09-01

    Technical Report 1315 Dissecting Situational Strength: Theoretical Analysis and Empirical Tests Reeshad S. Dalal George Mason ...George Mason University Charlie K. Brooks Georgia Institute of Technology September 2012 United States Army Research...MICHELLE SAMS, Ph.D. Director Research accomplished under contract for the Department of the Army George Mason University

  15. The emerging epidemic of methamphetamine-induced aortic dissections.

    PubMed

    Wako, Elizabeth; LeDoux, Denise; Mitsumori, Lee; Aldea, Gabriel S

    2007-01-01

    The clinical presentation, treatment, and outcomes of six consecutive patients presenting with acute aortic dissection secondary to hypertensive crises from methamphetamine use is described. Data were obtained prospectively from the expanded STS clinical database of the division of cardiothoracic surgery at the University of Washington, but reviewed in a retrospective fashion. These patients represent 5.5% of all patients diagnosed and treated for aortic dissection in the same time period (6/109) and 20% of all patients with aortic dissection under the age of 50 years (6/30). We conclude that young patients (dissections should be routinely tested for methamphetamine. Positive urine tests should be confirmed with chromatography-mass spectrometry (GC-MS). Beta and alpha blockers should be used instead of the more typical beta blockade alone. We recommend the addition and documentation of intense, long-term drug rehabilitation program along with routine periodic clinical and radiographic follow-up to prevent secondary aneurysmal dilation of remaining pathological aorta.

  16. Neurovascular Bundle Decompression without Excessive Dissection for Tarsal Tunnel Syndrome

    PubMed Central

    KIM, Kyongsong; ISU, Toyohiko; MORIMOTO, Daijiro; SASAMORI, Toru; SUGAWARA, Atsushi; CHIBA, Yasuhiro; ISOBE, Masahiro; KOBAYASHI, Shiro; MORITA, Akio

    2014-01-01

    Tarsal tunnel syndrome (TTS) is an entrapment neuropathy of the posterior tibial nerve and its branches in the tarsal tunnel. We present our less invasive surgical treatment of TTS in 69 patients (116 feet) and their clinical outcomes. The mean follow-up period was 64.6 months. With the patient under local anesthesia we use a microscope to perform sharp dissection of the flexor retinaculum and remove the connective tissues surrounding the posterior tibial nerve and vessels. To prevent postoperative adhesion and delayed neuropathy, decompression is performed to achieve symptom improvement without excessive dissection. Decompression is considered complete when the patient reports intraoperative symptom abatement and arterial pulsation is sufficient. The sensation of numbness and/or pain and of foreign substance adhesion was reduced in 92% and 95% of our patients, respectively. In self-assessments, 47 patients (68%) reported the treatment outcome as satisfactory, 15 (22%) as acceptable, and 7 (10%) were dissatisfied. Of 116 feet, 4 (3%) required re-operation, initial decompression was insufficient in 2 feet and further decompression was performed; in the other 2 feet improvement was achieved by decompression of the distal tarsal tunnel. Our surgical method involves neurovascular bundle decompression to obtain sufficient arterial pulsation. As we use local anesthesia, we can confirm symptom improvement intraoperatively, thereby avoiding unnecessary excessive dissection. Our method is simple, safe, and without detailed nerve dissection and it prevents postoperative adhesion. PMID:25367582

  17. Neurovascular bundle decompression without excessive dissection for tarsal tunnel syndrome.

    PubMed

    Kim, Kyongsong; Isu, Toyohiko; Morimoto, Daijiro; Sasamori, Toru; Sugawara, Atsushi; Chiba, Yasuhiro; Isobe, Masahiro; Kobayashi, Shiro; Morita, Akio

    2014-01-01

    Tarsal tunnel syndrome (TTS) is an entrapment neuropathy of the posterior tibial nerve and its branches in the tarsal tunnel. We present our less invasive surgical treatment of TTS in 69 patients (116 feet) and their clinical outcomes. The mean follow-up period was 64.6 months. With the patient under local anesthesia we use a microscope to perform sharp dissection of the flexor retinaculum and remove the connective tissues surrounding the posterior tibial nerve and vessels. To prevent postoperative adhesion and delayed neuropathy, decompression is performed to achieve symptom improvement without excessive dissection. Decompression is considered complete when the patient reports intraoperative symptom abatement and arterial pulsation is sufficient. The sensation of numbness and/or pain and of foreign substance adhesion was reduced in 92% and 95% of our patients, respectively. In self-assessments, 47 patients (68%) reported the treatment outcome as satisfactory, 15 (22%) as acceptable, and 7 (10%) were dissatisfied. Of 116 feet, 4 (3%) required re-operation, initial decompression was insufficient in 2 feet and further decompression was performed; in the other 2 feet improvement was achieved by decompression of the distal tarsal tunnel. Our surgical method involves neurovascular bundle decompression to obtain sufficient arterial pulsation. As we use local anesthesia, we can confirm symptom improvement intraoperatively, thereby avoiding unnecessary excessive dissection. Our method is simple, safe, and without detailed nerve dissection and it prevents postoperative adhesion.

  18. Deep dissection: motivating students beyond rote learning in veterinary anatomy.

    PubMed

    Cake, Martin A

    2006-01-01

    The profusion of descriptive, factual information in veterinary anatomy inevitably creates pressure on students to employ surface learning approaches and "rote learning." This phenomenon may contribute to negative perceptions of the relevance of anatomy as a discipline. Thus, encouraging deep learning outcomes will not only lead to greater satisfaction for both instructors and learners but may have the added effect of raising the profile of and respect for the discipline. Consideration of the literature reveals the broad scope of interventions required to motivate students to go beyond rote learning. While many of these are common to all disciplines (e.g., promoting active learning, making higher-order goals explicit, reducing content in favor of concepts, aligning assessment with outcomes), other factors are peculiar to anatomy, such as the benefits of incorporating clinical tidbits, "living anatomy," the anatomy museum, and dissection classes into a "learning context" that fosters deep approaches. Surprisingly, the 10 interventions discussed focus more on factors contributing to student perceptions of the course than on drastic changes to the anatomy course itself. This is because many traditional anatomy practices, such as dissection and museum-based classes, are eminently compatible with active, student-centered learning strategies and the adoption of deep learning approaches by veterinary students. Thus the key to encouraging, for example, dissection for deep learning ("deep dissection") lies more in student motivation, personal engagement, curriculum structure, and "learning context" than in the nature of the learning activity itself.

  19. Using a Dissecting Microscope in Teaching Introductory Chemistry.

    ERIC Educational Resources Information Center

    Winokur, Robert; Monroe, Manus

    1985-01-01

    To have students develop observational skills and acquire an excitement about chemistry, stereoscopic dissecting microscopes are used to observe the physical characteristics and chemical reactions of various substances. Several of these reactions (including dissolving potassium permanganate in deionized water and reactions between copper metal and…

  20. Gastric Wall Dissection as a Complication of Percutaneous Gastrostomy

    SciTech Connect

    Reimer, Wolfgang; Farres, Maria Teresa; Lammer, Johannes

    1996-04-15

    A percutaneous gastrostomy (PG) was complicated by gastric wall dissection and partial tube malposition. It occurred after tangential puncture along the greater curvature of the stomach which was performed in order to avoid an enlarged left lobe of the liver. To prevent this complication we recommend not using hydrophilic guidewires during PG.

  1. Analysis of clinical features of painless aortic dissection.

    PubMed

    Liu, Zhao-yu; Zou, Yuan-lin; Chai, Bo-lan; Zeng, He-song

    2014-08-01

    The clinical characteristics of painless aortic dissection were investigated in order to improve the awareness of diagnosis and treatment of atypical aortic dissection. The 482 cases of aortic dissection were divided into painless group and pain group, and the data of the two groups were retrospectively analyzed. The major clinical symptom was pain in 447 cases (92.74%), while 35 patients (7.26%) had no typical pain. The gender, age, hypertension, hyperlipidemia, diabetes, smoking and drinking history had no statistically significant differences between the two groups (P>0.05). The proportion of Stanford type A in painless group was significantly higher than that in pain group (48.57% vs. 21.03%, P=0.006). The incidence of unconsciousness in the painless group was significantly higher than that in the pain group (14.29% vs. 3.58%, P=0.011). The incidence of hypotension in painless group was significantly higher than that in pain group for 4.26 folds (P=0.01). Computed tomography angiography (CTA) examination revealed that the incidence of aortic arch involved in the painless group was significantly higher than that in the pain group (19.23% vs. 5.52%, P=0.019). It was concluded that the incidence of painless aortic dissection was higher in Stanford A type patients, commonly seen in the patients complicated with hypotension and unconsciousness. CTA examination revealed higher incidence of aortic arch involvement.

  2. Apathetic syndrome from carotid dissection: a dangerous condition

    PubMed Central

    Gerace, Carmela; Corsi, Fabio Massimo; Comanducci, Giovanna

    2013-01-01

    Isolated behavioural disturbances can mimic psychiatric diseases and delay diagnosis of acute brain disease. We reported the case of a patient with carotid dissection manifesting only with apathetic syndrome that was initially considered as a possible postpartum depression, causing a threatening diagnostic delay. PMID:24000207

  3. Standard methods for Apis mellifera anatomy and dissection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An understanding of the anatomy and functions of internal and external structures are fundamental to many studies on the honey bee Apis mellifera. Similarly, proficiency in dissection techniques is vital for many more complex procedures. In this paper, which is a prelude to the other papers of the C...

  4. There Is More to the Dissection of a Pig's Heart

    ERIC Educational Resources Information Center

    Lee, Yeung Chung

    2004-01-01

    The dissection of the mammalian heart in secondary biology classes need not be restricted to revealing the internal structure of the heart and its function. It could also be used to demonstrate other important aspects of blood circulation, including the blood supply to the heart itself as well as the causes and effects of coronary heart disease.…

  5. General Dissection of Female Ant Reproductive System and Brain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dissection of the reproductive system of ant workers and queens can be useful for answering many questions. Observations of ovarian status in both female castes can be used to identify relationships between other factors and the ovaries, determine whether an individual has laid eggs, and, with more ...

  6. Case report on vertebral artery dissection in mixed martial arts.

    PubMed

    Slowey, Michael; Maw, Graeme; Furyk, Jeremy

    2012-04-01

    A 41-year-old man presented to the ED with severe vertigo 2 days after a grappling injury while training in mixed martial arts. Imaging revealed a cerebellar infarct with complete occlusion of the right vertebral artery secondary to dissection. Management options are discussed as is the ongoing controversy regarding the safety of the sport.

  7. Professional parachuting: the risk of acute aortic dissection.

    PubMed

    Buchholz, Stefan; Quaden, René Bombien; Schmitz, Christoph; Überfuhr, Peter

    2011-09-01

    Acute aortic dissection is a rare disease, but if it occurs rapid diagnosis and therapy are needed. It is usually seen in elderly patients with long-term persistent arterial hypertension. In younger patients, it is mainly caused by congenital connective tissue disorders, such as Marfan syndrome, or by trauma. We present here a 34-year-old male patient with an acute type A aortic dissection. This patient was a professional parachutist and had carried out a large number of parachute jumps during his lifetime. He was admitted to the emergency department with acute chest pain. The symptoms were not related in time to a parachute jump. During a computed tomography scan, an aortic dissection was diagnosed. The patient was immediately referred to the operating room, and the ascending aorta was replaced by a conduit. After a regular postoperative course, the patient was discharged and recovered completely. Although acute aortic dissection is rare in young patients, it has to be considered in cases of acute chest pain. An immediate diagnosis and adequate therapy are essential to offer the patient a good clinical outcome and long-term survival.

  8. Timing formulas for dissection algorithms on vector computers

    NASA Technical Reports Server (NTRS)

    Poole, W. G., Jr.

    1977-01-01

    The use of the finite element and finite difference methods often leads to the problem of solving large, sparse, positive definite systems of linear equations. MACSYMA plays a major role in the generation of formulas representing the time required for execution of the dissection algorithms. The use of MACSYMA in the generation of those formulas is described.

  9. Marfan syndrome, dissecting aneurysm of the aorta, and pregnancy

    PubMed Central

    Moore, H. C.

    1965-01-01

    A patient with the Marfan syndrome died suddenly from aortic rupture and dissection in the early puerperium of her second pregnancy. Although the association of the Marfan syndrome and pregnancy is extremely rare, the case reported here being only the fifth on record, the concurrence of dissecting aneurysm or aortic dissection with pregnancy is more frequent. Furthermore it is accepted that aortic dissection in young women below the age of 40 is more common in the pregnant than those not pregnant. The cause of the enhancing effect of pregnancy is unknown but is thought to be endocrine since the stability of connective tissue can be influenced by hormones, particularly the sex steroids. An unusual feature of the present case is the florid inflammatory reaction in the adventitia of the aorta, not specifically related to pregnancy or to the Marfan syndrome, and it is assumed that in this patient the congenital defect of connective tissue assumed to be the basis of the Marfan syndrome is associated with an acute collagen change or necrosis, possibly illustrating a link between the heritable disorders of connective tissue and the diffuse collagen disease. Images PMID:14304236

  10. Endovascular treatment of spontaneous isolated abdominal aortic dissection

    PubMed Central

    Giribono, Anna Maria; Ferrara, Doriana; Spalla, Flavia; Narese, Donatella; Bracale, Umberto; Pecoraro, Felice; Bracale, Renata; del Guercio, Luca

    2016-01-01

    Isolated abdominal aortic dissection is a rare clinical disease representing only 1.3% of all dissections. There are a few case series reported in the literature. The causes of this pathology can be spontaneous, iatrogenic, or traumatic. Most patients are asymptomatic and symptoms are usually abdominal or back pain, while claudication and lower limb ischemia are rare. Surgical and endovascular treatment are two valid options with acceptable results. We herein describe nine cases of symptomatic spontaneous isolated abdominal aortic dissection, out of which four successfully were treated with an endovascular approach between July 2003 and July 2013. All patients were men, smokers, symptomatic (either abdominal or back pain or lower limb ischemia), with a history of high blood pressure, with a medical history negative for concomitant aneurysmatic dilatation or previous endovascular intervention. Diagnosis of isolated abdominal aortic dissection were established by contrast-enhanced computed tomography angiography (CTA) of the thoracic and abdominal aorta. All nine patients initially underwent medical treatment. In four symptomatic cases, non-responsive to medical therapy, bare-metal stents or stent grafts were successfully positioned. All patients completed a CTA follow-up of at least 12 months, during which they remained symptom-free. Endovascular management of this condition is associated with a high rate of technical success and a low mortality; therefore, it can be considered the treatment of choice when it is feasible. PMID:27994881

  11. A Novel Approach to the Dissection of the Human Knee

    ERIC Educational Resources Information Center

    Clemente, F. Richard; Fabrizio, Philip A.; Shumaker, Michael

    2009-01-01

    The knee is one of the most frequently injured joints of the human body with injuries affecting the general population and the athletic population of many age groups. Dissection procedures for the knee joint typically do not allow unobstructed visualization of the anterior cruciate or posterior cruciate ligaments without sacrificing the collateral…

  12. Exploring Dissections of Rectangles into Right-Angled Triangles

    ERIC Educational Resources Information Center

    Griffiths, Martin

    2013-01-01

    In this article we highlight how a simple classroom activity associated with the dissection of rectangles into right-angled triangles can lead on to a number of interesting explorations for students following a post-16 mathematics course. Several results connected with this construction are obtained, and some of the educational benefits of…

  13. Student Attitudes to Whole Body Donation Are Influenced by Dissection

    ERIC Educational Resources Information Center

    Cahill, Kevin C.; Ettarh, Raj R.

    2008-01-01

    Given the important role that anatomical dissection plays in the shaping of medical student attitudes to life and death, these attitudes have not been evaluated in the context of whole body donation for medical science. First year students of anatomy in an Irish university medical school were surveyed by questionnaire before and after the initial…

  14. The Influence of Emotion on Students' Performance in Dissection Exercises

    ERIC Educational Resources Information Center

    Holstermann, Nina; Grube, Dietmar; Bogeholz, Susanne

    2009-01-01

    This paper investigates the issue of how emotions such as disgust influence students' self-efficacy belief in terms of mastering a dissection task and also how these affect their interest in the biology of the heart. Following models of intrinsic motivation and the development of motivation, we expected disgust to negatively impact on students'…

  15. Exploring dissections of rectangles into right-angled triangles

    NASA Astrophysics Data System (ADS)

    Griffiths, Martin

    2013-09-01

    In this article we highlight how a simple classroom activity associated with the dissection of rectangles into right-angled triangles can lead on to a number of interesting explorations for students following a post-16 mathematics course. Several results connected with this construction are obtained, and some of the educational benefits of carrying out an investigation such as this are also discussed.

  16. Gene expression levels assessed by CA1 pyramidal neuron and regional hippocampal dissections in Alzheimer’s disease

    PubMed Central

    Ginsberg, Stephen D.; Alldred, Melissa J.; Che, Shaoli

    2011-01-01

    To evaluate molecular signatures of an individual cell type in comparison to the associated region relevant towards understanding the pathogenesis of Alzheimer’s disease (AD), CA1 pyramidal neurons and the surrounding hippocampal formation were microaspirated via laser capture microdissection (LCM) from neuropathologically confirmed AD and age-matched control (CTR) subjects as well as from wild type mouse brain using single population RNA amplification methodology coupled with custom-designed microarray analysis with real-time quantitative polymerase-chain reaction (qPCR) validation. CA1 pyramidal neurons predominantly displayed downregulation of classes of transcripts related to synaptic transmission in AD versus CTR. Regional hippocampal dissections displayed downregulation of several overlapping genes found in the CA1 neuronal population related to neuronal expression, as well as upregulation of select transcripts indicative of admixed cell types including glial-associated markers and immediate-early and cell death genes. Gene level distributions observed in CA1 neurons and regional hippocampal dissections in wild type mice paralleled expression mosaics seen in postmortem human tissue. Microarray analysis was validated in qPCR studies using human postmortem brain tissue and CA1 sector and regional hippocampal dissections obtained from a mouse model of AD/Down syndrome (Ts65Dn mice) and normal disomic (2N) littermates. Classes of transcripts that have a greater percentage of the overall hybridization signal intensity within single neurons tended to be genes related to neuronal communication. The converse was also found, as classes of transcripts such as glial-associated markers were under represented in CA1 pyramidal neuron expression profiles relative to regional hippocampal dissections. These observations highlight a dilution effect that is likely to occur in conventional regional microarray and qPCR studies. Thus, single population studies of specific

  17. Internal carotid artery dissection following chiropractic treatment in a pregnant woman with Systemic Lupus Erythematosus.

    PubMed

    Morton, Adam

    2012-12-19

    A case of internal carotid artery dissection in a pregnant woman with Systemic Lupus Erythematosus (SLE) immediately following chiropractic treatment is presented. The literature regarding complications of neck manipulation during pregnancy, spontaneous dissection of craniocervical arteries in pregnancy and the postpartum period, and dissection of craniocervical arteries in SLE are reviewed. To the best of the author's knowledge, this is the first case of carotid artery dissection following chiropractic treatment in a pregnant woman published in the literature.

  18. Internal carotid artery dissection following chiropractic treatment in a pregnant woman with Systemic Lupus Erythematosus

    PubMed Central

    2012-01-01

    A case of internal carotid artery dissection in a pregnant woman with Systemic Lupus Erythematosus (SLE) immediately following chiropractic treatment is presented. The literature regarding complications of neck manipulation during pregnancy, spontaneous dissection of craniocervical arteries in pregnancy and the postpartum period, and dissection of craniocervical arteries in SLE are reviewed. To the best of the author’s knowledge, this is the first case of carotid artery dissection following chiropractic treatment in a pregnant woman published in the literature. PMID:23254252

  19. Outcomes of a rotational dissection system in gross anatomy.

    PubMed

    Marshak, David W; Oakes, Joanne; Hsieh, Pei-Hsuan; Chuang, Alice Z; Cleary, Leonard J

    2015-01-01

    At the University of Texas Houston Medical School, a rotational dissection system was introduced to improve coordination between the Gross Anatomy and the Introduction to Clinical Medicine (ICM) courses. Six students were assigned to each cadaver and divided into two teams. For each laboratory, one team was assigned to dissect and the other to attend ICM or study independently. For the next laboratory, the assignments were reversed. At the start of the session, the team that had dissected previously spent 30 minutes teaching the other team. In 2012, the students were given three traditional practical examinations with 50 questions drawn equally from each laboratory. Students also completed three mid-course evaluations. There were no significant differences in overall performance between the two teams. Nevertheless, we wanted to determine how well individual students identified structures they had dissected compared with those they had not. For dissected structures, the mean percent correct was 80.0 ± 13.0 (mean ± standard deviation), and for undissected structures, it was 78.3 ± 14.1. The difference was small, but statistically significant (P = 0.0007). Although this result validated the concerns expressed by some students, it did not appear that a change in the system was justified. Students were generally enthusiastic about the opportunity to learn clinical skills in the first semester of medical school, and 91-96% of the students agreed that learning anatomy at the same time helped them understand the physical examination exercises in ICM.

  20. Perceived Disgust and Personal Experiences are Associated with Acceptance of Dissections in Schools

    ERIC Educational Resources Information Center

    Fancovicova, Jana; Prokop, Pavol; Leskova, Andrea

    2013-01-01

    Animal dissections are essential parts of anatomy/zoology courses, but their effectiveness is influenced by student attitudes and emotions. Here we examined attitudes toward dissections in 397 prospective biology teachers enrolling two Slovak universities. Perceived disgust of dissections negatively correlated with other attitudes toward…

  1. A Comparison of V-Frog[C] to Physical Frog Dissection

    ERIC Educational Resources Information Center

    Lalley, James P.; Piotrowski, Phillip S.; Battaglia, Barbara; Brophy, Keith; Chugh, Kevin

    2010-01-01

    The purpose of the present study was to examine and compare the effectiveness of virtual frog dissection using V-Frog[C] and physical frog dissection on learning, retention, and affect. Subjects were secondary students enrolled in year-long life science classes in a suburban high school (N=102). Virtual dissections were done with V-Frog[C], a…

  2. Biology Teachers' Dissection Practices and the Influences that Lead to Their Adoption: An Exploratory Research

    ERIC Educational Resources Information Center

    Milano, Regina Nicole

    2010-01-01

    The lack of resolution in the on-going animal dissection debate inspired this mixed methods study to identify Connecticut secondary biology teachers' dissection practices and the influences that lead to their adoption. Qualitative findings indicate past experiences, managing objections to dissection, school culture, goals of biology teaching and…

  3. An Investigative Alternative to Single-Species Dissection in the Introductory Biology Laboratory

    ERIC Educational Resources Information Center

    Carlin, Joel L.

    2010-01-01

    Dissections of single species (e.g., fetal pig) are a common student learning activity in introductory biology courses. Such dissections demonstrate location of anatomical parts and provide dissection practice but provide less opportunity for student critical thinking, numeracy and demonstration of the scientific method. A comparative anatomy lab…

  4. The First Cut Is the Deepest: Reflections on the State of Animal Dissection in Biology Education

    ERIC Educational Resources Information Center

    De Villiers, Rian; Monk, Martin

    2005-01-01

    In biology education, the study of structure has traditionally involved the use of dissection. Animal-rights campaigners have caused biology educators and learners to question the necessity of dissections. This study reviews the research evidence for the efficacy of alternatives to dissection and then turns to research evidence on attitudes to…

  5. [Redo surgery for residual distal dissection after the limited proximal aortic replacement for Stanford type A acute aortic dissection].

    PubMed

    Ogino, Hitoshi

    2013-07-01

    Redo surgery for residual distal dissection after the limited proximal aortic repair for Stanford type A acute aortic dissection remains challenging with some difficulties. In essence, redo aortic repair predominantly depends on the significantly dilated parts of the residual dissection. According to that, the strategy including median or lateral approach and 1 or 2 staged repair would be determined with careful consideration for patients' age and function of the vital organs such as brain, heart, lung, liver, and kidney. Generally, for relatively young and low-risk patients, an aggressive 1 stage repair of the entire arch to descending aorta through a left thoracotomy is feasible. Meanwhile, 2 stage repair is beneficial for elderly high-risk patients, which consists of the 1st total arch replacement with elephant trunk through a median sternotomy followed secondly by the open descending aortic repair through a lateral thoracotomy or recently-advanced less-invasive endovascular aortic repair. In the initial repair, more aggressive total arch replacement with elephant trunk or frozen elephant trunk might be another useful option to potentially prevent such troublesome behaviors of the residual dissecting aorta requiring redo surgery in the late stage.

  6. Analysis of dissected tissues with digital holographic microscopy: quantification of inflammation mediated tissue alteration, influence of sample preparation, and reliability of numerical autofocusing

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Lenz, Philipp; Bettenworth, Dominik; Krausewitz, Philipp; Domagk, Dirk; Ketelhut, Steffi

    2015-03-01

    Quantitative phase imaging with digital holographic microscopy (DHM) allows label-free imaging of tissue sections and quantification of the spatial refractive index distribution, which is of interest for applications in digital pathology. We show that DHM allows quantitative imaging of different layers in unstained tissue samples by detection of refractive index changes. In addition, we evaluate the automated refocussing feature of DHM for application on dissected tissues and could achieve highly reproducible holographic autofocusing for unstained and moderately stained samples. Finally, it is demonstrated that in human ulcerative colitis patients the average tissue refractive index is reduced significantly in all parts of the inflamed colonic wall in comparison to patients in remission.

  7. Lymph node dissection for Siewert II esophagogastric junction adenocarcinoma

    PubMed Central

    Duan, Xiao-Feng; Yue, Jie; Tang, Peng; Shang, Xiao-Bin; Jiang, Hong-Jing; Yu, Zhen-Tao

    2017-01-01

    Abstract The present study was aimed to investigate the application of right thansthoracic Ivor–Lewis (IL), left transthoracic (LTT), and left thoracoabdominal (LTA) approach in Siewert type II adenocarcinoma of esophagogastric junction (AEG). The data of 196 patients with Siewert type II AEG received surgical resection in our cancer center between January 2014 and April 2016 was retrospectively analyzed. Finally, 136 patients met the inclusion criteria were enrolled in the study and divided into the IL (47 cases), LTT (51 cases), and LTA group (38 cases). Clinical and short-term treatment effects were compared among the 3 groups. The patients with weight loss, diabetes, and heart disease increased in the LTT group (P = 0.054, P = 0.075, and P = 0.063, respectively). Operation time was significantly longest in the IL group (P < 0.001), but the amount of bleeding and tumor size did not significantly differ among the 3 groups (P = 0.176 and P = 0.228, respectively). The IL group had the significantly longest proximal surgical margin (P < 0.001) and most number of total (P < 0.001) and thoracic lymph nodes (P < 0.001) dissected. Both the IL and LTA groups had more abdominal lymph nodes dissected than the LTT group (P < 0.001). In general, the IL and LTT groups had the highest dissection rates of every station of thoracic (P < 0.05) and lower mediastinal lymph nodes (P < 0.05), respectively. The dissection rate of the paracardial, left gastric artery, and gastric lesser curvature lymph nodes did not differ significantly among the 3 groups (P > 0.05), but the dissection rate of the hepatic artery, splenic artery, and celiac trunk lymph nodes was significantly highest in the IL group (P < 0.05). Postoperative hospital stay, perioperative complications, and mortality did not differ significantly among the 3 groups (P > 0.05). Compared with the traditional left transthoracic approach, the Ivor–Lewis approach

  8. Bare Stent Implantation in Iatrogenic Dissecting Pseudoaneurysm of the Superior Mesenteric Artery

    SciTech Connect

    Kutlu, Ramazan Ara, Cengiz; Sarac, Kaya

    2007-02-15

    Iatrogenic arterial dissection leading to the development of dissecting pseudoaneurysms of the superior mesenteric artery (SMA) is a rare complication of angiography. Surgical and endovascular treatment options exist for this important condition. We report a case of bare stent implantation in dissecting pseudoaneurysm of the SMA that developed after angiography in a patient with acute mesenteric ischemia. Although it is rarely published, iatrogenic arterial dissection causing pseudoaneurysm can occur after diagnostic and interventional angiography. Bare stent implantation in dissecting pseudoaneurysm of the SMA could be an advantageous endovascular treatment option in selected cases due its to potential preservation of important side branches of the SMA.

  9. Secondary science classroom dissections: Informing policy by evaluating cognitive outcomes and exploring affective outcomes

    NASA Astrophysics Data System (ADS)

    Allspaw, Kathleen M.

    Animal protection organizations claim that dissection is pedagogically unsound and that it will cause students to lose respect for non-human animals. Science teacher organizations support curricula that teach respect for animal life and include dissection. Prior research compared dissection to dissection alternatives. Four of the six studies revealed no difference between groups on tests of cognitive outcomes. One study revealed that dissection was superior, and one revealed that the alternative was superior. No differences in attitudes toward science, dissection or school were found. Attitudes toward non-human animals were not measured. This study focused on the dissections of earthworms and frogs in middle and high school classrooms. Pre and post-tests of conceptual understanding revealed failing scores and no significant pre/post differences. Because these tests required critical thinking skills, and the dissection activities did not, it is difficult to determine if the poor performance on these tests indicates the inability of the students to think critically, and/or if it indicates the ineffectiveness of dissection. Further studies of dissections that focus on critical thinking would be necessary to make this distinction. Classroom observations, student written narratives, and student and adult interviews revealed mixed attitudes toward non-human animals. Student behaviors during dissection were similar to those behaviors exhibited during non-dissection activities. Most students and adults readily supported worm dissections while they expressed some trepidation about frog dissections. Students and adults universally expressed affection for their pets and opposed the use of their own pets for dissection/research. There was slight support for the use of dogs and cats for dissection/research, but only those students who expressed hate for cats said that they could dissect cats. None of the students or adults expressed a willingness to dissect dogs. Some students

  10. Single nucleotide polymorphisms linked to quantitative trait loci for grain quality traits in wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wheat (Triticum aestivum L.) grain quality traits that are controlled by quantitative traits loci (QTLs) define suitable growing areas and potential end-use products of a wheat cultivar. To dissect the QTLs for these traits including protein content (GPC), test weight (TW), single kernel characteriz...

  11. Genome-wide association mapping of quantitative resistance to sudden death syndrome in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sudden death syndrome (SDS) is a serious threat to soybean production that can be controlled by host plant resistance. To dissect the genetic architecture of quantitative resistance to the disease in soybean, two independent association panels of soybean elite cultivar, consisting of 392 and 300 uni...

  12. Mapping quantitative trait loci for plant adaptation and morphology traits in wheat using single nucleotide polymorphisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wheat (Triticum aestivum L.) morphological and adaptation-related traits that are controlled by quantitative traits loci (QTL) define potential growing areas of a wheat cultivar. To dissect the QTL for heading date (HD), lodging resistance (LR), shattering resistance (SR), cold tolerance (CT), plant...

  13. Bilateral Carotid Artery Dissection after High Impact Road Traffic Accident

    PubMed Central

    Srivastava, Ankur; Bradley, Marcus; Kelly, Michael

    2008-01-01

    A 58 year old man was involved in a high impact road traffic incident and was admitted for observation. Asymptomatic for the first 24 hours, he collapsed with symptoms and signs consistent with a cerebrovascular accident. Computed tomography angiogram (CTA) and Magnetic resonance angiogram (MRA) demonstrated bilateral internal carotid artery dissections and a left middle cerebral artery infarct. It was not considered appropriate to attempt stenting or other revascularistation. The patient was treated with heparin prior to starting warfarin. He made a partial recovery and was discharged to a rehabilitation facility. This case is a reminder of carotid dissection as an uncommon but serious complication of high speed motor vehicle accident, which may be silent initially. Literature Review suggests risk stratification before relevant radiological screening at risk patients. Significant advances in CTA have made it the diagnostic tool of choice, but ultrasound is an important screening tool. PMID:22470607

  14. Traumatic vertebral artery dissection presenting with incomplete congruous homonymous quadrantanopia

    PubMed Central

    2010-01-01

    Background To describe a rare presentation of vertebral artery dissection (VAD) as a small but congruous incomplete homonymous hemianopia demonstrating use of visual field testing in the diagnosis. Case presentation A 30 year old woman had been unwell for 4 months with difficulty focusing, vertigo, dizziness and a feeling of falling to the right. A small but congruous right inferior homonymous quadrantanopia was found on examination leading to further investigation that uncovered a vertebral artery dissection and multiple posterior circulation infarctions including a left occipital stroke matching the field defect. Conclusions We describe an atypical case of VAD presenting with a small congruous quadrantanopia. This is a rare but significant condition that predisposes to multiple thromboembolic infarction that may be easily misdiagnosed and a high index of suspicion is required to make the diagnosis. PMID:20482837

  15. Management of Acute Aortic Syndrome and Chronic Aortic Dissection

    SciTech Connect

    Nordon, Ian M. Hinchliffe, Robert J.; Loftus, Ian M.; Morgan, Robert A.; Thompson, Matt M.

    2011-10-15

    Acute aortic syndrome (AAS) describes several life-threatening aortic pathologies. These include intramural hematoma, penetrating aortic ulcer, and acute aortic dissection (AAD). Advances in both imaging and endovascular treatment have led to an increase in diagnosis and improved management of these often catastrophic pathologies. Patients, who were previously consigned to medical management or high-risk open surgical repair, can now be offered minimally invasive solutions with reduced morbidity and mortality. Information from the International Registry of Acute Aortic Dissection (IRAD) database demonstrates how in selected patients with complicated AAD the 30-day mortality from open surgery is 17% and endovascular stenting is 6%. Despite these improvements in perioperative deaths, the risks of stroke and paraplegia remain with endovascular treatment (combined outcome risk 4%). The pathophysiology of each aspect of AAS is described. The best imaging techniques and the evolving role of endovascular techniques in the definitive management of AAS are discussed incorporating strategies to reduce perioperative morbidity.

  16. [Stanford type a acute aortic dissection with pectus excavatum].

    PubMed

    Kuwata, Toshiyuki; Fukuda, Hirotsugu; Yoshitatsu, Masao; Yamada, Yasuyuki; Shibasaki, Ikuko; Inoue, Yuho; Hori, Takayuki; Ogawa, Hironaga; Tsuchiya, Go; Shimizu, Riha; Takei, Yusuke

    2012-11-01

    Pectus excavatum is generally an isolated abnormality of the chest wall. However, some patients have a concomitant pectus deformity and cardiac & aortic disease. Decisions must be made regarding the operative approach and whether the pectus excavatum should be corrected during the same session. We report 2 patients with acute Stanford type A aortic dissection and pectus excavatum who underwent emergency operation. In case 1, median sternotomy is an unsuitable approach for open heart surgery, since the heart and great vessels are displace into the left hemithorax. But combined sternotomy and left anterior thoracotomy provided excellent surgical exposure. In case 2, we proceeded with a leftsided costotomy of four ribs and place a normal chest retractor providing as excellent exposure as combined sternotomy and left anterior thoracotomy. A left-sided costotomy of four ribs can be performed safely, eliminating the risks of median sternotomy in acute stanford type A aortic dissection with pectus excavatum.

  17. High-energy drinks may provoke aortic dissection.

    PubMed

    Jonjev, Zivojin S; Bala, Gustav

    2013-05-01

    High-energy drinks have become extremely popular after Red Bull's promotion at 1987 in Austria and 1997 in the United States. Since then, we witnessed spectacular increase in different brands, caffeine content and market consumption all over the world. However, there are no reports published in the scientific literature related with detrimental side effects after heavy consumption of high-energy drinks. We report a series of three high-risk cardiovascular patients who had aortic dissection (De Bakey type I and II) following significant consumption of high-energy drinks. All of them required emergency surgical procedure and were remaining stable after surgery. We propose that uncontrolled consumption of high-energy drinks, especially in patients with underlying heart disease, could provoke potentially lethal cardiovascular events as well as acute aortic dissection.

  18. Mutations in Myosin Light Chain Kinase Cause Familial Aortic Dissections

    PubMed Central

    Wang, Li; Guo, Dong-chuan; Cao, Jiumei; Gong, Limin; Kamm, Kristine E.; Regalado, Ellen; Li, Li; Shete, Sanjay; He, Wei-Qi; Zhu, Min-Sheng; Offermanns, Stephan; Gilchrist, Dawna; Elefteriades, John; Stull, James T.; Milewicz, Dianna M.

    2010-01-01

    Mutations in smooth muscle cell (SMC)-specific isoforms of α-actin and β-myosin heavy chain, two major components of the SMC contractile unit, cause familial thoracic aortic aneurysms leading to acute aortic dissections (FTAAD). To investigate whether mutations in the kinase that controls SMC contractile function (myosin light chain kinase [MYLK]) cause FTAAD, we sequenced MYLK by using DNA from 193 affected probands from unrelated FTAAD families. One nonsense and four missense variants were identified in MYLK and were not present in matched controls. Two variants, p.R1480X (c.4438C>T) and p.S1759P (c.5275T>C), segregated with aortic dissections in two families with a maximum LOD score of 2.1, providing evidence of linkage of these rare variants to the disease (p = 0.0009). Both families demonstrated a similar phenotype characterized by presentation with an acute aortic dissection with little to no enlargement of the aorta. The p.R1480X mutation leads to a truncated protein lacking the kinase and calmodulin binding domains, and p.S1759P alters amino acids in the α-helix of the calmodulin binding sequence, which disrupts kinase binding to calmodulin and reduces kinase activity in vitro. Furthermore, mice with SMC-specific knockdown of Mylk demonstrate altered gene expression and pathology consistent with medial degeneration of the aorta. Thus, genetic and functional studies support the conclusion that heterozygous loss-of-function mutations in MYLK are associated with aortic dissections. PMID:21055718

  19. Pathophysiology and Surgical Treatment of Type A Acute Aortic Dissection

    PubMed Central

    Karube, Norihisa; Yasuda, Shota; Miyamoto, Takuma; Matsuki, Yusuke; Isoda, Susumu; Goda, Motohiko; Suzuki, Shinichi; Masuda, Munetaka; Imoto, Kiyotaka

    2016-01-01

    Objectives: We report the pathophysiology and treatment results of type A acute aortic dissection from our 20-year experience. Methods: We studied 673 patients with type A acute aortic dissection who underwent initial treatment from 1994 through July 2014. We divided these patients into two groups. The former group comprised 448 patients from 1994 through 2008, and the latter group comprised 225 patients from 2009 onward, when the current strategy of initial treatment and surgical technique including the early organ reperfusion therapies were established. Results: Women were significantly often presented than men in patients over 60 years of age. Thrombosed-type dissection accounted for more than half in patients over 70 years, and significantly often complicated pericardial effusion and cardiac tamponade than patent type. Malperfusion occurred in 26% of patients. Central repair operations were performed in 579 patients. In-hospital mortality for all patients was 15%, and for the patients who underwent central repair operations was 10%. Former period of operation, malperfusion, and preoperative cardiopulmonary arrest were significant risk factor of in-hospital death. Preoperative left main trunk (LMT) stents were placed in eight patients and superior mesenteric artery (SMA) intervention was performed in five, they were effective to improve the outcome. From 2009 onward, in-hospital mortality was 5.0% and there was no significant risk factor. Conclusion: Surgical results of type A acute aortic dissection were dramatically improved in the past 20 years. Early reperfusion strategy for the patients with malperfusion improved the outcomes. (This article is a translation of Jpn J Vasc Surg 2015; 24: 127–134.) PMID:27738456

  20. A sneeze: an unusual trigger for aortic dissection.

    PubMed

    Upadhyaya, Subramanya G N; Large, A

    2013-12-03

    We describe a rare case of type B thoracic aortic dissection resulting from a forceful sneeze in a 57-year-old man. The presenting clinical features were not typical and consisted of pleuritic chest pain and breathlessness following a forceful sneeze. There was no haemodynamic compromise. The diagnosis was made incidentally when he underwent a CT pulmonary angiography as part of pulmonary embolism work-up.

  1. Modeling plaque fissuring and dissection during balloon angioplasty intervention.

    PubMed

    Gasser, T Christian; Holzapfel, Gerhard A

    2007-05-01

    Balloon angioplasty intervention is traumatic to arterial tissue. Fracture mechanisms such as plaque fissuring and/or dissection occur and constitute major contributions to the lumen enlargement. However, these types of mechanically-based traumatization of arterial tissue are also contributing factors to both acute procedural complications and chronic restenosis of the treatment site. We propose physical and finite element models, which are generally useable to trace fissuring and/or dissection in atherosclerotic plaques during balloon angioplasty interventions. The arterial wall is described as an anisotropic, heterogeneous, highly deformable, nearly incompressible body, whereas tissue failure is captured by a strong discontinuity kinematics and a novel cohesive zone model. The numerical implementation is based on the partition of unity finite element method and the interface element method. The later is used to link together meshes of the different tissue components. The balloon angioplasty-based failure mechanisms are numerically studied in 3D by means of an atherosclerotic-prone human external iliac artery, with a type V lesion. Image-based 3D geometry is generated and tissue-specific material properties are considered. Numerical results show that in a primary phase the plaque fissures at both shoulders of the fibrous cap and stops at the lamina elastica interna. In a secondary phase, local dissections between the intima and the media develop at the fibrous cap location with the smallest thickness. The predicted results indicate that plaque fissuring and dissection cause localized mechanical trauma, but prevent the main portion of the stenosis from high stress, and hence from continuous tissue damage.

  2. The Chronobiology of Stanford Type A Aortic Dissections

    PubMed Central

    DeAnda, Abe; Grossi, Eugene A.; Balsam, Leora B.; Moon, Marc R.; Barlow, Clifford W.; Navia, Daniel O.; Ursomanno, Patricia; Ziganshin, Bulat A.; Rabinovich, Annette E.; Elefteriades, John A.; Smith, Julian A.

    2015-01-01

    Background Seasonal variations of Stanford Type A dissections (STADs) have been previously described in the Northern Hemisphere (NH). This study sought to determine if these variation are mirrored in the Southern Hemisphere (SH). Methods Data from patients treated surgically for STADs were retrospectively obtained from existing administrative and clinical databases from NH and SH sites. Data points of interest included age, sex, date of dissection, and 30-day mortality. The dates of dissections (independent of year) were then organized by season. Results A total of 1418 patients were identified (729 NH and 689 SH) with complete data available for 1415; 896 patients were male with a mean age was 61 ± 14 years, and the overall 30-day mortality was 17.3%. Comparison of NH and SH on a month-to-month basis demonstrated a 6-month phase shift and a significant difference by season, with STADs occurring predominantly in the winter and least in the summer. Decomposition of the monthly incidence using Fourier analysis revealed the phase shift of the primary harmonic to be –21.9 and 169.8 degrees (days), respectively, for NH and SH. The resultant 191.7 day difference did not exactly correspond to the anticipated 6-month difference but was compatible with the original hypothesis. Conclusion Chronobiology plays a role in the occurrence of STADs with the highest occurrence in the winter months independent of the hemisphere. Season is not the predominant reason why aortas dissect, but for patients at risk, the increase in systemic vascular resistance during the winter months may account for the seasonal variations seen. PMID:27390746

  3. Relict landscape resistance to dissection by upstream migrating knickpoints

    NASA Astrophysics Data System (ADS)

    Brocard, Gilles Y.; Willenbring, Jane K.; Miller, Thomas E.; Scatena, Frederik N.

    2016-06-01

    Expanses of subdued topographies are common at high elevation in mountain ranges. They are often interpreted as relict landscapes and are expected to be replaced by steeper topography as erosion proceeds. Preservation of such relict fragments can merely reflect the fact that it takes time to remove any preexisting topography. However, relict fragments could also possess intrinsic characteristics that make them resilient to dissection. We document here the propagation of a wave of dissection across an uplifted relict landscape in Puerto Rico. Using 10Be-26Al burial dating on cave sediments, we show that uplift started 4 Ma and that river knickpoints have since migrated very slowly across the landscape. Modern detrital 10Be erosion rates are consistent with these long-term rates of knickpoint retreat. Analysis of knickpoint distribution, combined with visual observations along the streambeds, indicates that incision by abrasion and plucking is so slow that bedrock weathering becomes a competing process of knickpoint retreat. The studied rivers flow over a massive stock of quartz diorite surrounded by an aureole of metavolcanic rocks. Earlier studies have shown that vegetation over the relict topography efficiently limits erosion, allowing for the formation of a thick saprolite underneath. Such slow erosion reduces streambed load fluxes delivered to the knickpoints, as well as bed load grain size. Both processes limit abrasion. Compounding the effect of slow abrasion, wide joint spacing in the bedrock makes plucking infrequent. Thus, the characteristics of the relict upstream landscape have a direct effect on stream incision farther downstream, reducing the celerity at which the relict, subdued landscape is dissected. We conclude that similar top-down controls on river incision rate may help many relict landscapes to persist amidst highly dissected topographies.

  4. Ventricular Septal Dissection Complicating Inferior Wall Myocardial Infarction

    PubMed Central

    Kalvin, Lindsey; Yousefzai, Rayan; Khandheria, Bijoy K.; Paterick, Timothy E.

    2017-01-01

    Postmyocardial infarction ventricular septal defect is an increasingly rare mechanical complication of acute myocardial infarction. We present a case of acute myocardial infarction from right coronary artery occlusion that developed hypotension and systolic murmur 12 hours after successful percutaneous coronary intervention. Although preoperative imaging suggested a large ventricular septal defect and a pseudoaneurysm, intraoperative findings concluded a serpiginous dissection of the ventricular septum. The imaging technicalities are discussed.

  5. Human Dissection in Medical Education: More than Just Anatomy.

    PubMed

    Rehkämper, Gerd

    2016-01-01

    The dissection course is an essential component of the medical curriculum. Nonetheless, the time expenditure and intensity of supervising the students in this course has been diminishing since the 1970s. That endangers not only the transmission of fundamental knowledge of anatomy. It also concerns key concepts such as establishing values, the concept of humans, and physician competencies, because medical education must be seen not merely as fact-directed instruction but instead should be connected with a mission for professional acculturation.

  6. Spontaneous Coronary Artery Dissection: Case Series from a Tertiary Centre.

    PubMed

    Anderson, Robert D; Jayadeva, Pavithra S; Wilson, William M; Iyer, Ravi

    2016-03-01

    Spontaneous coronary artery dissection (SCAD) is a rare cause of non-atherosclerotic acute coronary syndrome (ACS). As it is more commonly seen in young women, the diagnosis can be missed. Current evidence is based on case reports and retrospective studies with no consensus recommendations on immediate management and long-term follow-up. We present a case series of four patients to outline clinical presentation, prognosis and long-term management of this rare clinical entity.

  7. Imaging and Quantitation Techniques for Tracking Cargo along Endosome-to-Golgi Transport Pathways

    PubMed Central

    Chia, Pei Zhi Cheryl; Gleeson, Paul A.

    2013-01-01

    Recent improvements in the resolution of light microscopy, coupled with the development of a range of fluorescent-based probes, have provided new approaches to dissecting membrane domains and the regulation of membrane trafficking. Here, we review these advances, as well as highlight developments in quantitative image analysis and novel unbiased analytical approaches to quantitate protein localization. The application of these approaches to endosomal sorting and endosome-to-Golgi transport is discussed. PMID:24709647

  8. Endovascular Treatment of Iatrogenic and Traumatic Carotid Artery Dissection

    SciTech Connect

    Schulte, Stefan; Donas, Konstantinos P. Pitoulias, Georgios A.; Horsch, Svante

    2008-09-15

    This paper reports on the early and midterm results of endovascular treatment of acute carotid artery dissections, its specific problems, and its limitations. We encountered seven patients with symptomatic extracranial carotid artery dissection, three cases of which occurred after carotid endarterectomy, two after carotid angioplasty and stenting, and two after trauma. Balloon-expandable and self-expanding stents were placed using a transfemoral approach. Success in restoring the carotid lumen was achieved in all patients. No procedure-related complications occurred. All patients experienced significant clinical improvement while in the hospital and achieved complete long-term recovery. At follow-up (mean, 22.4 months), good luminal patency of the stented segments was observed. In conclusion, in this small series, primary stent-supported angioplasty seems to be a safe and effective strategy in the treatment of selected patients having acute traumatic extracranial carotid artery dissection, with excellent early and midterm results. Larger series and longer-term follow-up are required before definitive recommendations can be made.

  9. Update in the management of type B aortic dissection.

    PubMed

    Nauta, Foeke Jh; Trimarchi, Santi; Kamman, Arnoud V; Moll, Frans L; van Herwaarden, Joost A; Patel, Himanshu J; Figueroa, C Alberto; Eagle, Kim A; Froehlich, James B

    2016-06-01

    Stanford type B aortic dissection (TBAD) is a life-threatening aortic disease. The initial management goal is to prevent aortic rupture, propagation of the dissection, and symptoms by reducing the heart rate and blood pressure. Uncomplicated TBAD patients require prompt medical management to prevent aortic dilatation or rupture during subsequent follow-up. Complicated TBAD patients require immediate invasive management to prevent death or injury caused by rupture or malperfusion. Recent developments in diagnosis and management have reduced mortality related to TBAD considerably. In particular, the introduction of thoracic stent-grafts has shifted the management from surgical to endovascular repair, contributing to a fourfold increase in early survival in complicated TBAD. Furthermore, endovascular repair is now considered in some uncomplicated TBAD patients in addition to optimal medical therapy. For more challenging aortic dissection patients with involvement of the aortic arch, hybrid approaches, combining open and endovascular repair, have had promising results. Regardless of the chosen management strategy, strict antihypertensive control should be administered to all TBAD patients in addition to close imaging surveillance. Future developments in stent-graft design, medical therapy, surgical and hybrid techniques, imaging, and genetic screening may improve the outcomes of TBAD patients even further. We present a comprehensive review of the recommended management strategy based on current evidence in the literature.

  10. [Axillary lymph node dissection in clinically occult breast cancer].

    PubMed

    Le Bouëdec, G; Pomel, C; Chamussy, E; Feillel, V; de Latour, M; Dauplat, J

    1996-07-01

    The study concerns 265 patients with axillary lymph node dissection for non-palpable breast cancer. The mammographically detected breast tumors were: 36 ductal carcinomas in situ (DCIS), 23 microinvasive carcinomas, 206 invasive carcinomas of which 179 were invasive ductal cancers (IDC), 25 invasive lobular cancers (ILC) and 2 mucinous invasive carcinomas. The histologic size of the invasive component was < or = 5 mm in 38 cases, 6-10 mm in 84 cases, 11-15 mm in 53 cases, 16-20 mm in 16 cases, > 20 mm in 15 cases. Axillary dissection was performed immediately during the initial surgical procedure in 209 patients (79%) or secondarily in 56 (21%) according to the results of intraoperative examination of surgical specimens on frozen sections. Axillary lymph node involvement was not found in DCIS, microinvasive carcinomas or invasive carcinomas < or = 5 mm in size. Among all 206 invasive breast carcinomas, lymph node involvement was found in 7.8% (16/206) of cases. There were 9/84 (10.7%) in tumors > 10 mm, 7/122 (5.8%) in tumors < or = 10 mm. Thus, it is concluded that lymph node involvement is unlikely to be found in patients with non palpable breast cancers, specially those with carcinoma in situ, microinvasive breast tumors and invasive breast cancer with less than 5 mm maximum diameter size. Axillary dissection may be avoided in these patients. However, the use of new prognostic factors of lymph node involvement may help in the definition of patient group.

  11. Propagation of dissection in a residually-stressed artery model.

    PubMed

    Wang, Lei; Roper, Steven M; Hill, Nicholas A; Luo, Xiaoyu

    2017-02-01

    This paper studies dissection propagation subject to internal pressure in a residually-stressed two-layer arterial model. The artery is assumed to be infinitely long, and the resultant plane strain problem is solved using the extended finite element method. The arterial layers are modelled using the anisotropic hyperelastic Holzapfel-Gasser-Ogden model, and the tissue damage due to tear propagation is described using a linear cohesive traction-separation law. Residual stress in the arterial wall is determined by an opening angle [Formula: see text] in a stress-free configuration. An initial tear is introduced within the artery which is subject to internal pressure. Quasi-static solutions are computed to determine the critical value of the pressure, at which the dissection starts to propagate. Our model shows that the dissection tends to propagate radially outwards. Interestingly, the critical pressure is higher for both very short and very long tears. The simulations also reveal that the inner wall buckles for longer tears, which is supported by clinical CT scans. In all simulated cases, the critical pressure is found to increase with the opening angle. In other words, residual stress acts to protect the artery against tear propagation. The effect of residual stress is more prominent when a tear is of intermediate length ([Formula: see text]90[Formula: see text] arc length). There is an intricate balance between tear length, wall buckling, fibre orientation, and residual stress that determines the tear propagation.

  12. Talking about Death: Implementing Peer Discussion as a Coping Mechanism to Overcome Fears about Dissection, Death, and Dying

    ERIC Educational Resources Information Center

    Kotze, Sanet Henriet; Mole, Calvin Gerald

    2013-01-01

    Many studies have reported on the perceptions of medical students toward dissection. It is important to understand the feelings and symptoms experienced during dissection so that they can be adequately handled. Prior to dissection, first year students are given lectures on aspects of dissection, death and dying, and death rituals in various…

  13. Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation

    PubMed Central

    Lee, Joomin; Ahn, Eunsook; Park, Wyun Kon; Park, Seyeon

    2016-01-01

    Inhalation anesthetics are used to decrease the spinal cord transmission of painful stimuli. However, the molecular or biochemical processes within cells that regulate anesthetic-induced responses at the cellular level are largely unknown. Here, we report the phosphoproteome profile of SH-SY5y human neuroblastoma cells treated with sevoflurane, a clinically used anesthetic. Phosphoproteins were isolated from cell lysates and analyzed using two-dimensional gel electrophoresis. The phosphorylation of putative anesthetic-responsive marker proteins was validated using western blot analysis in cells treated with both sevoflurane and isoflurane. A total of 25 phosphoproteins were identified as differentially phosphorylated proteins. These included key regulators that signal cytoskeletal remodeling steps in pathways related to vesicle trafficking, axonal growth, and cell migration. These proteins included the Rho GTPase, Ras-GAP SH3 binding protein, Rho GTPase activating protein, actin-related protein, and actin. Sevoflurane and isoflurane also resulted in the dissolution of F-actin fibers in SH-SY5y cells. Our results show that anesthetics affect the phosphorylation of proteins involved in cytoskeletal remodeling pathways. PMID:27611435

  14. Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration.

    PubMed

    Chen, Yanling; Lu, Bingwen; Yang, Qingkai; Fearns, Colleen; Yates, John R; Lee, Jiing-Dwan

    2009-04-15

    Integrins interact with extracellular matrix (ECM) and deliver intracellular signaling for cell proliferation, survival, and motility. During tumor metastasis, integrin-mediated cell adhesion to and migration on the ECM proteins are required for cancer cell survival and adaptation to the new microenvironment. Using stable isotope labeling by amino acids in cell culture-mass spectrometry, we profiled the phosphoproteomic changes induced by the interactions of cell integrins with type I collagen, the most common ECM substratum. Integrin-ECM interactions modulate phosphorylation of 517 serine, threonine, or tyrosine residues in 513 peptides, corresponding to 357 proteins. Among these proteins, 33 key signaling mediators with kinase or phosphatase activity were subjected to small interfering RNA-based functional screening. Three integrin-regulated kinases, DBF4, PAK2, and GRK6, were identified for their critical role in cell adhesion and migration possibly through their regulation of actin cytoskeleton arrangement. Altogether, we not only depict an integrin-modulated phosphorylation network during cell-ECM protein interactions but also reveal novel regulators for cell adhesion and migration.

  15. Classic Ras Proteins Promote Proliferation and Survival Via Distinct Phosphoproteome Alterations in Neurofibromin-Null Malignant Peripheral Nerve Sheath Tumor Cells

    PubMed Central

    Brossier, Nicole M.; Prechtl, Amanda M.; Longo, Jody Fromm; Barnes, Stephen; Wilson, Landon S.; Byer, Stephanie J.; Brosius, Stephanie N.; Carroll, Steven L.

    2015-01-01

    Neurofibromin, the tumor suppressor encoded by the neurofibromatosis type 1 (NF1) gene, potentially suppresses the activation of H-Ras, N-Ras and K-Ras. However, it is not known whether these classic Ras proteins are hyperactivated in NF1-null nerve sheath tumors, how they contribute to tumorigenesis and what signaling pathways mediate their effects. Here we show that H-Ras, N-Ras and K-Ras are coexpressed with their activators, (guanine nucleotide exchange factors), in neurofibromin-null malignant peripheral nerve sheath tumor (MPNST) cells and that all 3 Ras proteins are activated. Dominant negative (DN) H-Ras, a pan-inhibitor of the classic Ras family, inhibited MPNST proliferation and survival, but not migration. However, NF1-null MPNST cells were variably dependent on individual Ras proteins. In some lines, ablation of H-Ras, N-Ras and/or K-Ras inhibited mitogenesis. In others, ablation of a single Ras protein had no effect on proliferation; in these lines, ablation of a single Ras protein resulted in compensatory increases in the activation and/or expression of other Ras proteins. Using mass spectrometry-based phosphoproteomics, we identified 7 signaling networks affecting morphology, proliferation and survival that are regulated by DN H-Ras. Thus, neurofibromin loss activates multiple classic Ras proteins that promote proliferation and survival by regulating several distinct signaling cascades. PMID:25946318

  16. Phosphoproteomic Analyses of NRAS(G12) and NRAS(Q61) Mutant Melanocytes Reveal Increased CK2α Kinase Levels in NRAS(Q61) Mutant Cells.

    PubMed

    Posch, Christian; Sanlorenzo, Martina; Vujic, Igor; Oses-Prieto, Juan A; Cholewa, Brian D; Kim, Sarasa T; Ma, Jeffrey; Lai, Kevin; Zekhtser, Mitchell; Esteve-Puig, Rosaura; Green, Gary; Chand, Shreya; Burlingame, Alma L; Panzer-Grümayer, Renate; Rappersberger, Klemens; Ortiz-Urda, Susana

    2016-10-01

    In melanoma, mutant and thereby constantly active neuroblastoma rat sarcoma (NRAS) affects 15-20% of tumors, contributing to tumor initiation, growth, invasion, and metastasis. Recent therapeutic approaches aim to mimic RAS extinction by interfering with critical signaling pathways downstream of the mutant protein. This study investigates the phosphoproteome of primary human melanocytes bearing mutations in the two hot spots of NRAS, NRAS(G12) and NRAS(Q61). Stable isotope labeling by amino acids in cell culture followed by mass spectrometry identified 14,155 spectra of 3,371 unique phosphopeptides mapping to 1,159 proteins (false discovery rate < 2%). Data revealed pronounced PI3K/AKT signaling in NRAS(G12V) mutant cells and pronounced mitogen-activated protein kinase (MAPK) signaling in NRAS(Q61L) variants. Computer-based prediction models for kinases involved, revealed that CK2α is significantly overrepresented in primary human melanocytes bearing NRAS(Q61L) mutations. Similar differences were found in human NRAS(Q61) mutant melanoma cell lines that were also more sensitive to pharmacologic CK2α inhibition compared with NRAS(G12) mutant cells. Furthermore, CK2α levels were pronounced in patient samples of NRAS(Q61) mutant melanoma at the mRNA and protein level. The preclinical findings of this study reveal that codon 12 and 61 mutant NRAS cells have distinct signaling characteristics that could allow for the development of more effective, mutation-specific treatment modalities.

  17. [Late reoperations after repaired Stanford type A aortic dissection].

    PubMed

    Huang, F H; Li, L P; Su, C H; Qin, W; Xu, M; Wang, L M; Jiang, Y S; Qiu, Z B; Xiao, L Q; Zhang, C; Shi, H W; Chen, X

    2017-04-01

    Objective: To summarize the experience of reoperations on patients who had late complications related to previous aortic surgery for Stanford type A dissection. Methods: From August 2008 to October 2016, 14 patients (10 male and 4 female patients) who underwent previous cardiac surgery for Stanford type A aortic dissection accepted reoperations on the late complications at Department of Thoracic and Cardiovascular Surgery, Nanjing Hospital Affiliated to Nanjing Medical University. The range of age was from 41 to 76 years, the mean age was (57±12) years. In these patients, first time operations were ascending aorta replacement procedure in 3 patients, ascending aorta combined with partial aortic arch replacement in 4 patients, aortic root replacement (Bentall) associated with Marfan syndrome in 3 patients, aortic valve combined with ascending aorta replacement (Wheat) in 1 patient, ascending aorta combined with Sun's procedure in 1 patient, Wheat combined with Sun's procedure in 1 patient, Bentall combined with Sun's procedure in 1 patient. The interval between two operations averaged 0.3 to 10.0 years with a mean of (4.8±3.1) years. The reasons for reoperations included part anastomotic split, aortic valve insufficiency, false aneurysm formation, enlargement of remant aortal and false cavity. The selection of reoperation included anastomotic repair, aortic valve replacement, total arch replacement and Sun's procedure. Results: Of the 14 patients, the cardiopulmonary bypass times were 107 to 409 minutes with a mean of (204±51) minutes, cross clamp times were 60 to 212 minutes with a mean of (108±35) minutes, selective cerebral perfusion times were 16 to 38 minutes with a mean of (21±11) minutes. All patients survived from the operation, one patient died from severe pulmonary infection 50 days after operation. Three patients had postoperative complications, including acute renal failure of 2 patients and pulmonary infection of 1 patient, and these patients were

  18. Dissection as Inquiry: Using the "Peanut Observation" Activity to Promote a Revised Paradigm of Dissection and Facilitate Student Involvement and Understanding.

    ERIC Educational Resources Information Center

    Bernstein, Penny L.

    2000-01-01

    Introduces the peanut observation activity to teach about the pros and cons of dissection. As an inquiry-based approach, dissection is one way to teach process skills. Lists the progression of the activity as observation, questioning and finding the answer, challenge, discussion, and further examination. (Contains 12 references.) (YDS)

  19. Are All Hands-On Activities Equally Effective? Effect of Using Plastic Models, Organ Dissections, and Virtual Dissections on Student Learning and Perceptions

    ERIC Educational Resources Information Center

    Lombardi, Sara A.; Hicks, Reimi E.; Thompson, Katerina V.; Marbach-Ad, Gili

    2014-01-01

    This study investigated the impact of three commonly used cardiovascular model-assisted activities on student learning and student attitudes and perspectives about science. College students enrolled in a Human Anatomy and Physiology course were randomly assigned to one of three experimental groups (organ dissections, virtual dissections, or…

  20. Use of multimedia technology to provide solutions to existing curriculum problems: Virtual frog dissection

    NASA Astrophysics Data System (ADS)

    Youngblut, Christine

    The objective of this research was to determine whether currently available multimedia technology can resolve existing problems in the K--12 science curriculum. There are several practical and ethical problems relating to the classroom use of animal dissection and this led to the selection of hands-on frog dissection as the curriculum activity where the use of multimedia was investigated. The major finding was that multimedia-based virtual dissection was more effective than hands-on dissection in helping students learn about frog anatomy. Moreover, this result was achieved when the time available for the virtual dissection was approximately 44% less than that available for hands-on dissection. Examination of possible relationships between student characteristics and achievement revealed that students' attitudes to educational uses of animal dissection and their computer experience were positively correlated with their achievement scores. No relationships were found between either student gender or dissection experience and achievement test outcomes. Students rated virtual dissection as the easier of the two types of dissection, though they gave equivalent ratings for their enjoyment of virtual and hands-on dissection. Despite favorable feedback on the virtual dissection, a significant majority of students stated that they felt they would be "missing-out" on a valuable experience if they were not given the opportunity to perform a hands-on frog dissection. Comparing how students spent their time during each type of dissection showed that students spent a significantly larger proportion of their time On-Task when using the multimedia-based virtual dissection. In particular, the average increase in the proportion of time spent on activities directly related to the subject matter was over 36%. Time spent On-Task had a significant positive relationship with achievement for hands-on dissection. It may play a similar role for achievement with virtual dissection, but the

  1. [Dissecting aneurysms at the bases of the brachiocephalic artery and the left common carotid artery due to localized dissection of the aortic arch; report of a case].

    PubMed

    Higashi, Shigeki; Yoshida, Y; Mitsuoka, H

    2007-07-01

    A 43-year-old male lost consciousness immediately after archery practice, and was brought to our hospital by ambulance. Angiography showed dissecting aneurysms at the bases of the brachiocephalic artery and the left common carotid artery, causing compression of these arteries. Under cardiopulmonary bypass with selective cerebral perfusion, the blood supply to these arteries was restored with a bifurcated graft. Surgical specimen showed localized dissection of the aortic arch at the bifurcation to the brachiocephalic artery and the left common carotid artery, with the formation of dissecting aneurysms at the bases of both arteries. The aneurysms were filled with thrombi. In addition to these dissecting aneurysms, there were arterial dissections involving the brachiocephalic artery and the bilateral common carotid arteries. Histopathological examination of the vessel wall showed no evidence of atherosclerosis or vasculitis, and no abnormalities in the arrangement of elastic fibers.

  2. Acute aortic dissections with pregnancy in women with ACTA2 mutations.

    PubMed

    Regalado, Ellen S; Guo, Dong-chuan; Estrera, Anthony L; Buja, L Maximilian; Milewicz, Dianna M

    2014-01-01

    Mutations in ACTA2 predispose to thoracic aortic aneurysms and dissection as well as coronary artery and cerebrovascular disease. Here we examined the risk of aortic dissections, stroke and myocardial infarct with pregnancy in women with ACTA2 mutations. Of the 53 women who had a total of 137 pregnancies, eight had aortic dissections in the third trimester or the postpartum period (6% of pregnancies). One woman also had a myocardial infarct that occurred during pregnancy that was independent of her aortic dissection. Compared to the population-based frequency of peripartum aortic dissections of 0.6%, the rate of peripartum aortic dissections in women with ACTA2 mutations is much higher (8 out of 39; 20%). Six of these dissections initiated in the ascending aorta (Stanford type A), three were fatal. Three women had ascending aortic dissections at diameters less that 5.0 cm (range 3.8-4.7 cm). Aortic pathology showed mild to moderate medial degeneration of the aorta in three women. Of note, five of the women had hypertension either during or before the pregnancy. In summary, the majority of women with ACTA2 mutations did not have aortic or other vascular complications with pregnancy. However, these findings show that pregnancy is associated with significant risk for aortic dissection in women with ACTA2 mutations. Women with ACTA2 mutations who are planning to get pregnant should be counseled about this risk of aortic dissection, and proper clinical management should be initiated to reduce this risk.

  3. Diagnostic implication of fibrin degradation products and D-dimer in aortic dissection

    PubMed Central

    Dong, Jian; Duan, Xianli; Feng, Rui; Zhao, Zhiqing; Feng, Xiang; Lu, Qingsheng; Jing, Qing; Zhou, Jian; Bao, Junmin; Jing, Zaiping

    2017-01-01

    Fibrin degradation products (FDP) and D-dimer have been considered to be involved in many vascular diseases. In this study we aimed to explore the diagnostic implication of FDP and D-dimer in aortic dissection patients. 202 aortic dissection patients were collected as the case group, 150 patients with other cardiovascular diseases, including myocardial infarction (MI, n = 45), pulmonary infarction (n = 51) and abdominal aortic aneurysm (n = 54) were collected as non-dissection group, and 27 healthy people were in the blank control group. The FDP and D-dimer levels were detected with immune nephelometry. Logist regression analysis was performed to evaluate the influence of FDP and D-dimer for the aortic dissection patients. ROC curve was used to determine the diagnostic value of FDP and D-dimer. The FDP and D-dimer levels were significantly higher in aortic dissection patients than in non-dissection patients and the healthy controls. FDP and D-dimer were both the risk factors for patients with aortic dissection. From the ROC analysis, diagnostic value of FDP and D-dimer were not high to distinguish aortic dissection patients from the non-dissection patients. However FDP and D-dimer could be valuable diagnostic marker to differentiate aortic dissection patients and healthy controls with both AUC 0.863. PMID:28262748

  4. Diagnostic implication of fibrin degradation products and D-dimer in aortic dissection

    NASA Astrophysics Data System (ADS)

    Dong, Jian; Duan, Xianli; Feng, Rui; Zhao, Zhiqing; Feng, Xiang; Lu, Qingsheng; Jing, Qing; Zhou, Jian; Bao, Junmin; Jing, Zaiping

    2017-03-01

    Fibrin degradation products (FDP) and D-dimer have been considered to be involved in many vascular diseases. In this study we aimed to explore the diagnostic implication of FDP and D-dimer in aortic dissection patients. 202 aortic dissection patients were collected as the case group, 150 patients with other cardiovascular diseases, including myocardial infarction (MI, n = 45), pulmonary infarction (n = 51) and abdominal aortic aneurysm (n = 54) were collected as non-dissection group, and 27 healthy people were in the blank control group. The FDP and D-dimer levels were detected with immune nephelometry. Logist regression analysis was performed to evaluate the influence of FDP and D-dimer for the aortic dissection patients. ROC curve was used to determine the diagnostic value of FDP and D-dimer. The FDP and D-dimer levels were significantly higher in aortic dissection patients than in non-dissection patients and the healthy controls. FDP and D-dimer were both the risk factors for patients with aortic dissection. From the ROC analysis, diagnostic value of FDP and D-dimer were not high to distinguish aortic dissection patients from the non-dissection patients. However FDP and D-dimer could be valuable diagnostic marker to differentiate aortic dissection patients and healthy controls with both AUC 0.863.

  5. Hemodynamic Impact of a Spontaneous Cervical Dissection on an Ipsilateral Saccular Aneurysm

    PubMed Central

    See, Alfred P.; Penn, David L.; Du, Rose; Frerichs, Kai U

    2016-01-01

    The dynamic, hemodynamic impact of a cervical dissection on an ipsilateral, intracranial saccular aneurysm has not been well illustrated. This 45-year-old female was found to have a small, supraclinoid aneurysm ipsilateral to a spontaneous cervical internal carotid artery dissection. With healing of the dissection, the aneurysm appeared to have significantly enlarged. Retrospective review of the magnetic resonance imaging (MRI) at the time of the initial dissection demonstrated thrombus, similar in overall morphology to the angiographic appearance of the "enlarged" aneurysm. As the dissection healed far proximal to the intradural portion of the internal carotid artery, this suggested that the aneurysm was likely a typical, saccular posterior communicating artery aneurysm that had thrombosed and then recanalized secondary to flow changes from the dissection. The aneurysm was coiled uneventfully, in distinction from more complex treatment approaches such as flow diversion or proximal occlusion to treat an enlarging, dissecting pseudoaneurysm. This case illustrates that flow changes from cervical dissections may result in thrombosis of downstream saccular aneurysms. With healing, these aneurysms may recanalize and be misidentified as enlarging dissecting pseudoaneurysms. Review of an MRI from the time of the dissection facilitated the conclusion that the aneurysm was a saccular posterior communicating artery aneurysm, influencing treatment approach. PMID:27790401

  6. Human cadaveric dissection: a historical account from ancient Greece to the modern era.

    PubMed

    Ghosh, Sanjib Kumar

    2015-09-01

    The review article attempts to focus on the practice of human cadaveric dissection during its inception in ancient Greece in 3rd century BC, revival in medieval Italy at the beginning of 14th century and subsequent evolution in Europe and the United States of America over the centuries. The article highlights on the gradual change in attitude of religious authorities towards human dissection, the shift in the practice of human dissection being performed by barber surgeons to the anatomist himself dissecting the human body and the enactment of prominent legislations which proved to be crucial milestones during the course of the history of human cadaveric dissection. It particularly emphasizes on the different means of procuring human bodies which changed over the centuries in accordance with the increasing demand due to the rise in popularity of human dissection as a tool for teaching anatomy. Finally, it documents the rise of body donation programs as the source of human cadavers for anatomical dissection from the second half of the 20th century. Presently innovative measures are being introduced within the body donation programs by medical schools across the world to sensitize medical students such that they maintain a respectful, compassionate and empathetic attitude towards the human cadaver while dissecting the same. Human dissection is indispensable for a sound knowledge in anatomy which can ensure safe as well as efficient clinical practice and the human dissection lab could possibly be the ideal place to cultivate humanistic qualities among future physicians in the 21st century.

  7. Human cadaveric dissection: a historical account from ancient Greece to the modern era

    PubMed Central

    2015-01-01

    The review article attempts to focus on the practice of human cadaveric dissection during its inception in ancient Greece in 3rd century BC, revival in medieval Italy at the beginning of 14th century and subsequent evolution in Europe and the United States of America over the centuries. The article highlights on the gradual change in attitude of religious authorities towards human dissection, the shift in the practice of human dissection being performed by barber surgeons to the anatomist himself dissecting the human body and the enactment of prominent legislations which proved to be crucial milestones during the course of the history of human cadaveric dissection. It particularly emphasizes on the different means of procuring human bodies which changed over the centuries in accordance with the increasing demand due to the rise in popularity of human dissection as a tool for teaching anatomy. Finally, it documents the rise of body donation programs as the source of human cadavers for anatomical dissection from the second half of the 20th century. Presently innovative measures are being introduced within the body donation programs by medical schools across the world to sensitize medical students such that they maintain a respectful, compassionate and empathetic attitude towards the human cadaver while dissecting the same. Human dissection is indispensable for a sound knowledge in anatomy which can ensure safe as well as efficient clinical practice and the human dissection lab could possibly be the ideal place to cultivate humanistic qualities among future physicians in the 21st century. PMID:26417475

  8. Global quantitative analysis of phosphorylation underlying phencyclidine signaling and sensorimotor gating in the prefrontal cortex

    PubMed Central

    McClatchy, Daniel B.; Savas, Jeffrey N.; Martínez-Bartolomé, Salvador; Park, Sung Kyu; Maher, Pamela; Powell, Susan B.; Yates, John R.

    2015-01-01

    Prepulse inhibition (PPI) is an example of sensorimotor gating and deficits in PPI have been demonstrated in schizophrenia patients. Phencyclidine (PCP) suppression of PPI in animals has been studied to elucidate the pathological elements of schizophrenia. However, the molecular mechanisms underlying PCP treatment or PPI in the brain are still poorly understood. In this study, quantitative phosphoproteomic analysis was performed on the prefrontal cortex from rats that were subjected to PPI after being systemically injected with PCP or saline. PCP down-regulated phosphorylation events were significantly enriched in proteins associated with long-term potentiation (LTP). Importantly, this dataset identifies functionally novel phosphorylation sites on known LTP-associated signaling molecules. In addition, mutagenesis of a significantly altered phosphorylation site on xCT (SLC7A11), the light chain of system xc-, the cystine/glutamate antiporter, suggests that PCP also regulates the activity of this protein. Finally, new insights were also derived on PPI signaling independent of PCP treatment. This is the first quantitative phosphorylation proteomic analysis providing new molecular insights into sensorimotor gating. PMID:25869802

  9. Global quantitative analysis of phosphorylation underlying phencyclidine signaling and sensorimotor gating in the prefrontal cortex.

    PubMed

    McClatchy, D B; Savas, J N; Martínez-Bartolomé, S; Park, S K; Maher, P; Powell, S B; Yates, J R

    2016-02-01

    Prepulse inhibition (PPI) is an example of sensorimotor gating and deficits in PPI have been demonstrated in schizophrenia patients. Phencyclidine (PCP) suppression of PPI in animals has been studied to elucidate the pathological elements of schizophrenia. However, the molecular mechanisms underlying PCP treatment or PPI in the brain are still poorly understood. In this study, quantitative phosphoproteomic analysis was performed on the prefrontal cortex from rats that were subjected to PPI after being systemically injected with PCP or saline. PCP downregulated phosphorylation events were significantly enriched in proteins associated with long-term potentiation (LTP). Importantly, this data set identifies functionally novel phosphorylation sites on known LTP-associated signaling molecules. In addition, mutagenesis of a significantly altered phosphorylation site on xCT (SLC7A11), the light chain of system xc-, the cystine/glutamate antiporter, suggests that PCP also regulates the activity of this protein. Finally, new insights were also derived on PPI signaling independent of PCP treatment. This is the first quantitative phosphorylation proteomic analysis providing new molecular insights into sensorimotor gating.

  10. The core of a competent surgeon: a working knowledge of surgical anatomy and safe dissection techniques.

    PubMed

    Rogers, Robert M; Taylor, Richard H

    2011-12-01

    The competent gynecologic surgeon has a sure, working knowledge of the anatomy in the field of pelvic dissection and is expert in the techniques and in the millimeter by millimeter progression of surgical dissections. When operating in the pelvis, the surgeon always asks several questions. The first is, “In what anatomic area am I dissecting?” This question defines the anatomy to be dissected out. The second is,“What dissection techniques will I use here?” The measured steps of surgical dissection give the surgeon the confidence to proceed with the operation, while safeguarding the integrity of the surrounding anatomic structures. With less blood loss and less trauma to the tissues and anatomic structures, the surgeon may expect a better surgical outcome for the patient.

  11. Dissecting repulsion linkage in the dwarfing gene Dw3 region for sorghum plant height provides insights into heterosis.

    PubMed

    Li, Xin; Li, Xianran; Fridman, Eyal; Tesso, Tesfaye T; Yu, Jianming

    2015-09-22

    Heterosis is a main contributor to yield increase in many crop species. Different mechanisms have been proposed for heterosis: dominance, overdominance, epistasis, epigenetics, and protein metabolite changes. However, only limited examples of molecular dissection and validation of these mechanisms are available. Here, we present an example of discovery and validation of heterosis generated by a combination of repulsion linkage and dominance. Using a recombinant inbred line population, a separate quantitative trait locus (QTL) for plant height (qHT7.1) was identified near the genomic region harboring the known auxin transporter Dw3 gene. With two loci having repulsion linkage between two inbreds, heterosis in the hybrid can appear as a single locus with an overdominance mode of inheritance (i.e., pseudo-overdominance). Individually, alleles conferring taller plant height exhibited complete dominance over alleles conferring shorter height. Detailed analyses of different height components demonstrated that qHT7.1 affects both the upper and lower parts of the plant, whereas Dw3 affects only the part below the flag leaf. Computer simulations show that repulsion linkage could influence QTL detection and estimation of effect in segregating populations. Guided by findings in linkage mapping, a genome-wide association study of plant height with a sorghum diversity panel pinpointed genomic regions underlying the trait variation, including Dw1, Dw2, Dw3, Dw4, and qHT7.1. Multilocus mixed model analysis confirmed the advantage of complex trait dissection using an integrated approach. Besides identifying a specific genetic example of heterosis, our research indicated that integrated molecular dissection of complex traits in different population types can enable plant breeders to fine tune the breeding process for crop production.

  12. Dissecting repulsion linkage in the dwarfing gene Dw3 region for sorghum plant height provides insights into heterosis

    PubMed Central

    Li, Xin; Li, Xianran; Fridman, Eyal; Tesso, Tesfaye T.; Yu, Jianming

    2015-01-01

    Heterosis is a main contributor to yield increase in many crop species. Different mechanisms have been proposed for heterosis: dominance, overdominance, epistasis, epigenetics, and protein metabolite changes. However, only limited examples of molecular dissection and validation of these mechanisms are available. Here, we present an example of discovery and validation of heterosis generated by a combination of repulsion linkage and dominance. Using a recombinant inbred line population, a separate quantitative trait locus (QTL) for plant height (qHT7.1) was identified near the genomic region harboring the known auxin transporter Dw3 gene. With two loci having repulsion linkage between two inbreds, heterosis in the hybrid can appear as a single locus with an overdominance mode of inheritance (i.e., pseudo-overdominance). Individually, alleles conferring taller plant height exhibited complete dominance over alleles conferring shorter height. Detailed analyses of different height components demonstrated that qHT7.1 affects both the upper and lower parts of the plant, whereas Dw3 affects only the part below the flag leaf. Computer simulations show that repulsion linkage could influence QTL detection and estimation of effect in segregating populations. Guided by findings in linkage mapping, a genome-wide association study of plant height with a sorghum diversity panel pinpointed genomic regions underlying the trait variation, including Dw1, Dw2, Dw3, Dw4, and qHT7.1. Multilocus mixed model analysis confirmed the advantage of complex trait dissection using an integrated approach. Besides identifying a specific genetic example of heterosis, our research indicated that integrated molecular dissection of complex traits in different population types can enable plant breeders to fine tune the breeding process for crop production. PMID:26351684

  13. Gastric cancer: Current status of lymph node dissection

    PubMed Central

    Degiuli, Maurizio; De Manzoni, Giovanni; Di Leo, Alberto; D’Ugo, Domenico; Galasso, Erica; Marrelli, Daniele; Petrioli, Roberto; Polom, Karol; Roviello, Franco; Santullo, Francesco; Morino, Mario

    2016-01-01

    D2 procedure has been accepted in Far East as the standard treatment for both early (EGC) and advanced gastric cancer (AGC) for many decades. Recently EGC has been successfully treated with endoscopy by endoscopic mucosal resection or endoscopic submucosal dissection, when restricted or extended Gotoda's criteria can be applied and D1+ surgery is offered only to patients not fitted for less invasive treatment. Furthermore, two randomised controlled trials (RCTs) have been demonstrating the non inferiority of minimally invasive technique as compared to standard open surgery for the treatment of early cases and recently the feasibility of adequate D1+ dissection has been demonstrated also for the robot assisted technique. In case of AGC the debate on the extent of nodal dissection has been open for many decades. While D2 gastrectomy was performed as the standard procedure in eastern countries, mostly based on observational and retrospective studies, in the west the Medical Research Council (MRC), Dutch and Italian RCTs have been conducted to show a survival benefit of D2 over D1 with evidence based medicine. Unfortunately both the MRC and the Dutch trials failed to show a survival benefit after the D2 procedure, mostly due to the significant increase of postoperative morbidity and mortality, which was referred to splenopancreatectomy. Only 15 years after the conclusion of its accrual, the Dutch trial could report a significant decrease of recurrence after D2 procedure. Recently the long term survival analysis of the Italian RCT could demonstrate a benefit for patients with positive nodes treated with D2 gastrectomy without splenopancreatectomy. As nowadays also in western countries D2 procedure can be done safely with pancreas preserving technique and without preventive splenectomy, it has been suggested in several national guidelines as the recommended procedure for patients with AGC. PMID:26973384

  14. LOX Mutations Predispose to Thoracic Aortic Aneurysms and Dissections

    PubMed Central

    Guo, Dong-chuan; Regalado, Ellen S.; Gong, Limin; Duan, Xueyan; Santos-Cortez, Regie Lyn P.; Arnaud, Pauline; Ren, Zhao; Cai, Bo; Hostetler, Ellen M.; Moran, Rocio; Liang, David; Estrera, Anthony; Safi, Hazim J; Leal, Suzanne M.; Bamshad, Michael J.; Shendure, Jay; Nickerson, Deborah A.; Jondeau, Guillaume; Boileau, Catherine; Milewicz, Dianna M.

    2016-01-01

    Rationale Mutations in several genes have been identified that are responsible for approximately 25% of families with familial thoracic aortic aneurysms and dissections (TAAD). However, the causative gene remains unknown in 75% of families. Objectives To identify the causative mutation in families with autosomal dominant inheritance of TAAD. Methods and Results Exome sequencing was used to identify the mutation responsible for a large family with TAAD. A heterozygous rare variant, c.839G>T (p.Ser280Arg), was identified in LOX, encoding a lysyl oxidase, that segregated with disease in the family. Sanger and exome sequencing was performed to investigate mutations in candidate genes in an additional 410 probands from unrelated families. Additional LOX rare variants that segregated with disease in families were identified, including c.125G>A (p.Trp42*), c.604G>T (p.Gly202*), c.743C>T (p.Thr248Ile), c.800A>C (p.Gln267Pro), and c.1044T>A (p.Ser348Arg). The altered amino acids cause haploinsufficiency for LOX or are located at a highly conserved LOX catalytic domain, which is relatively invariant in the population. Expression of the LOX variants p.Ser280Arg and p.Ser348Arg had significantly lower lysyl oxidase activity when compared with the wild type protein. Individuals with LOX variants had fusiform enlargement of the root and ascending thoracic aorta, leading to ascending aortic dissections. Conclusions These data, along with previous studies showing the deficiency of LOX in mice or inhibition of lysyl oxidases in turkeys and rats causes aortic dissections, support the conclusion that rare genetic variants in LOX predispose to thoracic aortic disease. PMID:26838787

  15. Clinical Characteristics of Symptomatic Vertebral Artery Dissection. A Systematic Review